Adsorption of immunopotentiators to insoluble metal salts

ABSTRACT

Immunopotentiators can be adsorbed to insoluble metal salts, such as aluminium salts, to modify their pharmacokinetics, pharmacodynamics, intramuscular retention time, and/or immunostimulatory effect. Immunopotentiators are modified to introduce a moiety, such as a phosphonate group, which can mediate adsorption. These modified compounds can retain or improve their in vivo immunological activity even when delivered in an adsorbed form.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/820,370, with an international filing date of Sep. 1, 2011, now U.S.Pat. No. 9,315,530, which is the National Stage of International PatentApplication No. PCT/US2011/050231, filed Sep. 1, 2011, which claims thebenefit of U.S. Provisional Application Nos. 61/379,126, filed Sep. 1,2010: 61/448,394, filed Mar. 2, 2011: and 61/466,887, filed Mar. 23,2011, the disclosures of which are hereby incorporated by reference intheir entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 303822015710SeqList.txt,date recorded: Apr. 5, 2016, size: 25 KB).

TECHNICAL FIELD

The invention is in the field of formulating immunopotentiatingcompounds for in vivo use. More particularly, the invention relates tothe design of immunopotentiating agents for formulation by associationwith insoluble metal salts e.g. by adsorption.

BACKGROUND ART

Early detection of specific classes of pathogens is accomplished by theinnate immune system with help of pattern recognition receptors (PRRs).The detected pathogens include viruses, bacteria, protozoa and fungi,and each constitutively expresses a set of class-specific,mutation-resistant molecules called pathogen-associated molecularpatterns (PAMPs).

Toll-like receptors (TLRs) are an important family of PRRs and arewidely expressed on innate immune cells, including dendritic cells(DCs), macrophages, mast cells, neutrophils, endothelial cells andfibroblasts. TLRs have broad specificity for conserved molecularpatterns shared by bacteria, viruses and parasites.

A number of different TLRs have been characterized. These TLRs bind andbecome activated by different ligands, which in turn are located ondifferent organisms or structures. The development of immunopotentiatorcompounds that are capable of eliciting responses in specific TLRs is ofinterest in the art.

For example, reference I describes certain lipopeptide molecules thatare TLR2 agonists. References 2 to 5 each describe classes of smallmolecule agonists of TLR7. References 6 & 7 describe TLR7 and TLR8agonists for treatment of diseases. These various compounds includesmall molecule immunopotentiators (SMIPs).

These compounds have typically been selected for their TLR-modulatingactivity, without attention to their pharmacokinetic or pharmacodynamic(PK/PD) properties or for their retention at an injection site wherethey can usefully exert an immunostimulatory effect. The compounds maythus have poor PK/PD or retention profiles when introduced in vivo.Moreover, it may be desirable to modify the systemic exposure of thecompounds e.g. to minimise potential systemic side effects in widespreadprophylactic immunisations, or to maintain systemic exposure in anemergency immunotherapeutic setting. As shown in FIGS. 1 to 5, injectionof a TLR7 active SMIP into a subject initially results in high serumconcentration which quickly drops, and injected compounds can be fullycleared from muscle within 24 hours.

It is an object of the invention to provide new formulations ofimmunopotentiators, and in particular of SMIPs, which can modify orimprove the immunopotentiators' pharmacological properties, such asPK/PD profiles, cellular uptake, retention at injection sites, reducedgeneral activation of B cells, etc. It is a further object to provideformulations which can improve their immunostimulating activity

DISCLOSURE OF THE INVENTION

The inventors have surprisingly found that the PK/PD ofimmunopotentiators (and in particular TLR agonists), and their retentionat sites of injection, can be modified by adsorbing them to insolublemetal salts, such as aluminium salts. Stable adsorption of the compoundsideally takes place by ligand exchange, but known SMIPs typically lacksuitable functional groups. Thus SMIPs can be modified to introduce anadsorptive moiety, such as a phosphonate group, which can then mediateadsorption. The inventors have found that these modified SMIPs (inparticular, TLR7 agonists) can retain their in vivo immunologicalactivity even when delivered in an adsorbed form, and so the improvedPK/PD properties are not at the expense of immunostimulating activity.Indeed, adsorption of TLR7 agonists is shown herein to improve theirimmunostimulatory activity. Furthermore, adsorption of the compounds canreduce peak serum concentrations and increase residence times at sitesof intramuscular injection, which can contribute to modifying andcontrolling the level of systemic exposure. High systemic exposure canelicit the production of high levels of proinflammatory cytokines in theblood, so higher residence time at an injection site can help tominimise the production of proinflammatory cytokines in the blood, thusimproving safety and/or tolerability of the compounds. Cellular uptakeof the compounds can also be enhanced by adsorption.

The invention enables the modification of SMIPs to contain at least oneadsorptive moiety such that the modified SMIP has the ability to adsorbto insoluble metal salt adjuvants. The inventors have realised the broadapplicability of this modification and subsequent formulation forimmunopotentiating agents as a whole. Therefore, described herein are abroad range of functionalised immunopotentiating compounds andcompositions comprising these functionalised compounds that areillustrative of the present invention.

In a first aspect, the invention provides a TLR agonist comprising atleast one adsorptive moiety, with the proviso that the TLR agonist:

-   -   (a) is not a TLR4 agonist;    -   (b) is not a TLR9 agonist;    -   (c) is not a compound according to formula (I) or (II) as        defined below;    -   (d) is not a compound according to formula (III) as defined        below;    -   (e) is not a compound according to Formula (I-A) as defined        below.

The presence of the adsorptive moiety enables the TLR agonist to beformulated with an insoluble metal salt adjuvant in order to modify orimprove its pharmacokinetic and pharmacodynamic profile, to increase theduration of its retention at a site of intramuscular injection, and/orto increase its immunostimulatory effect.

In a second aspect, the invention provides a water-soluble TLR agonistcomprising at least one adsorptive moiety, with the proviso that thewater-soluble TLR agonist:

-   -   (a) is not a TLR4 agonist;    -   (b) is not a TLR9 agonist;    -   (c) is not a compound according to formula (I) or (II) as        defined below; and    -   (d) is not a compound according to formula (III) as defined        below.

In a third aspect, the invention provides a TLR agonist comprising atleast one phosphonate group, with the proviso that the TLR agonist:

-   -   (a) is not a phosphonate-containing compound according to        formula (I) or (II) as defined below;    -   (b) is not a phosphonate-containing compound according to        formula (III), as defined below.    -   (c) is not a phosphonate-containing compound according to        formula (I-A), as defined below.

In a fourth aspect, the invention provides a compound comprising atleast one adsorptive moiety, wherein the compound is an agonist of TLR1,TLR3, TLR5, TLR6, TLR8 or TLR11. The invention also provides such acompound adsorbed to an insoluble metal salt. Thus the inventionprovides a composition comprising the agonist compound and an insolublemetal salt, wherein the agonist compound is adsorbed onto the insolublemetal salt.

In a fifth aspect, the invention provides a compound comprising at leastone adsorptive moiety, wherein the compound is an agonist of TLR1, TLR2,TLR3, TLR5, TLR6, TLR7, TLR8, or TLR11, with the proviso that thecompound:

-   -   (a) is not a compound according to formula (I) or (II) as        defined below;    -   (b) is not a compound according to formula (III), as defined        below; and    -   (c) is not a compound according to formula (I-A), as defined        below.

In a sixth aspect, the invention provides a composition comprising a TLRagonist and an insoluble metal salt, wherein the TLR agonist is adsorbedonto the insoluble metal salt, with the proviso that the TLR agonist:

-   -   (a) is not a TLR4 agonist;    -   (b) is not a TLR9 agonist;    -   (c) is not a compound according to formula (II) as defined        below; and    -   (d) is not a compound according to Formula (I-A) as defined        below.

In some embodiments of this sixth aspect, the compound does not includean acyl chain or a cytosine nucleotide. In some embodiments of thissixth aspect, the agonist compound has a molecular weight below 1500 Dae.g. below 1300 Da, or preferably below 1000 Da.

According to a seventh aspect, the invention provides a compositioncomprising a TLR agonist and an insoluble metal salt, wherein the TLRagonist is adsorbed onto the insoluble metal salt, with the proviso thatthe TLR agonist:

-   -   (a) is not a compound according to formula (III) as defined        below;    -   (b) is not a compound according to Formula (I-A) as defined        below;    -   (c) does not include an acyl chain; and    -   (d) does not include a cytosine nucleotide.

Preferably, agonists of this seventh aspect have a molecular weightbelow 1000 Da.

According to an eighth aspect, the invention provides a TLR4 agonistcomprising at least one adsorptive moiety, provided that the TLR4agonist does not include an acyl chain. The invention also provides sucha TLR4 agonist adsorbed to an insoluble metal salt. Thus the inventionprovides a composition comprising the TLR4 agonist compound and aninsoluble metal salt, wherein the TLR4 agonist compound is adsorbed ontothe insoluble metal salt.

According to a ninth aspect, the invention provides a TLR9 agonistcomprising at least one adsorptive moiety, provided that the TLR9agonist does not include a cytidine nucleoside. The invention alsoprovides such a TLR9 agonist adsorbed to an insoluble metal salt. Thusthe invention provides a composition comprising the TLR9 agonistcompound and an insoluble metal salt, wherein the TLR9 agonist compoundis adsorbed onto the insoluble metal salt.

According to a tenth aspect, the invention provides a TLR2 agonistcomprising at least one adsorptive moiety, provided that the TLR2agonist is not a compound according to formula (III) as defined below.The invention also provides such a TLR2 agonist adsorbed to an insolublemetal salt. Thus the invention provides a composition comprising theTLR2 agonist compound and an insoluble metal salt, wherein the TLR2agonist compound is adsorbed onto the insoluble metal salt.

According to an eleventh aspect, the invention provides a TLR7 agonistcomprising at least one adsorptive moiety, provided that the TLR7agonist is not: (a) a compound according to formula (I) as definedbelow; (b) a compound according to formula (II) as defined below; or (c)a compound according to formula (I-A) as defined below. The inventionalso provides such a TLR7 agonist adsorbed to an insoluble metal salt.Thus the invention provides a composition comprising the TLR7 agonistcompound and an insoluble metal salt, wherein the TLR7 agonist compoundis adsorbed onto the insoluble metal salt.

According to a twelfth aspect, the invention provides a TLR4 agonistcomprising at least one adsorptive moiety, wherein the TLR agonist has amolecular weight of less than 1000 Da. The invention also provides sucha TLR4 agonist adsorbed to an insoluble metal salt. Thus the inventionprovides a composition comprising the TLR4 agonist compound and aninsoluble metal salt, wherein the TLR4 agonist compound is adsorbed ontothe insoluble metal salt.

According to a thirteenth aspect, the invention provides a TLR9 agonistcomprising at least one adsorptive moiety, wherein the TLR agonist has amolecular weight of less than 1000 Da. The invention also provides sucha TLR9 agonist adsorbed to an insoluble metal salt. Thus the inventionprovides a composition comprising the TLR9 agonist compound and aninsoluble metal salt, wherein the TLR9 agonist compound is adsorbed ontothe insoluble metal salt.

According to a fourteenth aspect, the invention provides a TLR agonistincluding between two and fifteen adsorptive moieties. In someembodiments of this aspect, the TLR agonist is (i) not a TLR4 agonist(ii) not a TLR9 agonist (iii) not a compound of formula (III) and/or(iv) not a compound of formula (IV). The invention also provides such anagonist adsorbed to an insoluble metal salt. Thus the invention providesa composition comprising the agonist compound and an insoluble metalsalt, wherein the agonist compound is adsorbed onto the insoluble metalsalt.

According to a fifteenth aspect, the invention provides a compositioncomprising a TLR agonist, an insoluble metal salt, and a buffer, whereinthe TLR agonist is adsorbed onto the insoluble metal salt, with theproviso that (i) the buffer is not a phosphate buffer. In someembodiments of this aspect, the TLR agonist is (i) not a TLR4 agonist(ii) not a TLR9 agonist (iii) not a compound of formula (III) and/or(iv) not a compound of formula (IV).

According to a sixteenth aspect, the invention provides a TLR7 agonistselected from compounds according to any of formulae (C), (D), (E), and(H):

wherein:

-   -   (a) P³ is selected from H, C₁-C₆alkyl, CF₃, and        —((CH₂)_(p)O)_(q)(CH₂)_(p)O₈— and —Y-L-X—P(O)(OR^(X))(OR^(Y));        and P⁴ is selected from H, C₁-C₆alkyl, —C₁-C₆alkylaryl and        —Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one        of P³ and P⁴ is —Y-L-X—P(O)(OR^(X))(OR^(Y)),    -   (b) P³ is selected from H, C₁-C₆alkyl, and        —Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁶ is selected from H, C₁-C₆alkyl        each optionally substituted with 1 to 3 substituents selected        from C₁-C₄alkyl and OH, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁷        is selected from H, C₁-C₆alkyl,        —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)—, —NHC₁-C₆alkyl and        —Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one        of P⁵, P⁶ and P⁷ is —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   (c) P⁸ is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        —NHC₁-C₆alkyl each optionally substituted with OH, and        —Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁹ and P¹⁰ are each        independently selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        —NHC₁-C₆alkyl each optionally substituted with OH and        C₁-C₆alkyl, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso        that at least one of P⁸, P⁹ or P¹⁰ is        —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   (d) P¹⁶ and each P¹⁸ are each independently selected front H,        C₁-C₆alkyl, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹⁷ is selected        from H, C₁-C₆alkyl, aryl, heteroaryl, C₁-C₆alkylaryl, C₁-C₆alkyl        heteroaryl, C₁-C₆alkylaryl-Y-L-X—P(O)(OR^(X))(OR^(Y)) and        —Y-L-X—P(O)(OR^(X))(OR^(Y)), each optionally substituted with 1        to 2 substituents selected from C₁-C₆alkyl or heterocyclyl with        the proviso that at least one of P¹⁶, P¹⁷ or a P¹⁸ contains a        —Y-L-X—P(O)(OR^(X))(OR^(Y)) moiety;    -   R^(X) and R^(Y) are independently selected from H and        C₁-C₆alkyl;    -   R^(C), R^(D) and R^(H) are each independently selected from H        and C₁-C₆alkyl;    -   X^(C) is selected from CH and N;    -   R^(E) is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,        C(O)C₁-C₆alkyl, halogen and —((CH₂)_(p)O)_(q)(CH₂)_(p)—;    -   X^(E) is selected from a covalent bond, CR^(E2)R^(E3) and        NR^(E4);    -   R^(E2), R^(E3) and R^(E4) are independently selected from H and        C₁-C₆alkyl;    -   X^(H1)-X^(H2) is selected from —CR^(H2)R^(H3)—,        —CR^(H2)R^(H3)—CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—,        —C(O)CR^(H2)R^(H3)—, —CR^(H2)R^(H3)C(O)—, —NR^(H4)C(O)—,        C(O)NR^(H4)—, CR^(H2)R^(H3)S(O)₂ and —CR^(H2)═CR^(H2)—;    -   R^(H2), R^(H3) and R^(H4) are each independently selected from        H, C₁-C₆alkyl and P¹⁸;    -   X^(H3) is selected from N and CN;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)— each optionally substituted with 1 to 4        substituents independently selected from halo, OH, C₁-C₄alkyl,        —OP(O)(OH)₂ and —P(O)(OH)₂;    -   m is selected from 0 or 1;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6;    -   q is selected from 1, 2, 3 and 4; and    -   s is selected from 0 and 1.

According to a seventeenth aspect, the invention provides a TLR8 agonistselected from compounds according to formula (G):

wherein:

-   -   P¹¹ is selected from H, C₁-C₆alkyl, C₁-C₆ alkoxy, NR^(V)R^(W)        and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   P¹² is selected from H, C₁-C₆alkyl, aryl optionally substituted        by —C(O)NR^(V)R^(W), and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   P¹³, P¹⁴ and P¹⁵ are independently selected from H, C₁-C₆alkyl,        C₁-C₆ alkoxy and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   with the proviso that at least one of P¹¹, P¹², P¹³, P¹⁴ or P¹⁵        is —Y-L-X—P(O)(OR^(X))(OR^(Y));

R^(V) and R^(W) are independently selected from H, C₁-C₆alkyl ortogether with the nitrogen atom to which they are attached form a 4 to 7remembered heterocyclic ring;

-   -   X^(G) is selected from C, CH and N;    -   represents an optional double bond, wherein X^(G) is C if        is a double bond; and    -   R^(G) is selected from H and C₁-C₆alkyl;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene,    -   C₁-C₆alkyleneoxy and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally        substituted with 1 to 4 substituents independently selected from        halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6 and    -   q is selected from 1, 2, 3 and 4.

According to an eighteenth aspect, the invention provides a compound offormulae (B) or (F) or (J):

-   -   as defined below.

The seventeenth and eighteenth aspects include such agonists of formulae(B) or (G) adsorbed to an insoluble metal salt. Thus the inventionprovides a composition comprising the agonist compound and an insolublemetal salt, wherein the agonist compound is adsorbed onto the insolublemetal salt.

According to a nineteenth aspect, the invention provides a process forpreparing an adjuvant complex, comprising a step of mixing a TLR agonistwith an insoluble metal salt such that the TLR agonist adsorbs to theinsoluble metal salt to form the complex. The TLR agonist is preferablya TLR agonist as variously described above e.g. in some embodiments theagonist is not a TLR4 agonist, a TLR9 agonist, a compound of formula(III), or a compound of formula (I-A). The invention also provides anadjuvant complex obtained or obtainable by this process. The complex canbe mixed with an immunogen to provide an immunogenic composition.

According to a twentieth aspect, the invention provides a process forpreparing a sterile adjuvant complex, comprising steps of: (i) mixing aTLR agonist with an insoluble metal salt such that the TLR agonistadsorbs to the insoluble metal salt to form the complex; and (ii)sterilising the complex. The agonist is preferably an agonist asvariously described above e.g. in some embodiments the agonist is not aTLR4 agonist or a TLR9 agonist. The invention also provides a sterileadjuvant complex obtained or obtainable by this process. The sterilecomplex can be mixed with an immunogen to provide an immunogeniccomposition. Sterilisation can be conveniently achieved by autoclaving(or similar procedures [8]).

The invention also provides a process for preparing a sterile adjuvantcomplex, comprising steps of: (i) sterilising a solution or suspensionof a TLR agonist; and (ii) combining the sterilised solution orsuspension with a sterile insoluble metal salt. The invention alsoprovides a process for preparing a sterile adjuvant complex, comprisingsteps of: (i) sterilising an insoluble metal salt; and (ii) combiningthe sterilised insoluble metal salt with a sterile solution orsuspension of a TLR agonist. The invention also provides a process forpreparing a sterile adjuvant complex, comprising a step of combining asterile solution or suspension of a TLR agonist with a sterile insolublemetal salt. Sterilisation of the TLR agonist solution/suspension canconveniently be achieved by sterile filtration, and this material can beprepared in concentrated form. Sterilisation of the insoluble metal saltcan conveniently be achieved by autoclaving. The sterile insoluble metalsalt will typically be an aqueous suspension.

According to a twenty-first aspect, the invention provides a process forpreparing an immunogenic composition, wherein the process comprisesmixing a TLR agonist, an insoluble metal salt, and an immunogen, therebyproviding the immunogenic composition. The invention also provides animmunogenic composition obtained or obtainable by this process. In someembodiments of this aspect, the TLR agonist is not compound 13 herein.In some embodiments of this aspect, the immunogen is not a three-proteinmixture of 287-953, 936-741 and 961c as disclosed in references 40 & 73.In some embodiments of this aspect, the TLR agonist is (i) not a TLR4agonist (ii) not a TLR9 agonist (iii) not a compound of formula (III)and/or (iv) not a compound of formula (IV).

According to a twenty-second aspect, the invention provides a processfor preparing an immunogenic composition, comprising one of: (i)combining an immunogen with a mixture comprising a TLR agonist and aninsoluble metal salt; (ii) combining an insoluble metal salt with amixture comprising a TLR agonist and an immunogen; or (iii) combining aTLR agonist with a mixture comprising an insoluble metal salt and animmunogen. The invention also provides an immunogenic compositionobtained or obtainable by this process. In some embodiments of thisaspect, the TLR agonist is not compound 13 herein. In some embodimentsof this aspect, the immunogen is not a three-protein mixture of 287-953,936-741 and 961c as disclosed in references 40 & 73. In some embodimentsof this aspect, the TLR agonist is (i) not a TLR4 agonist (ii) not aTLR9 agonist (iii) not a compound of formula (III) and/or (iv) not acompound of formula (IV).

According to a twenty-third aspect, the invention provides a method formodifying or improving the pharmacokinetic profile of animmunopotentiator, and/or for increasing the time for which animmunopotentiator is retained at a site of intramuscular injection, themethod comprising: chemically modifying the immunopotentiator to form amodified immunopotentiator by the introduction of an adsorptive group.The adsorptive group may be joined to the immunopotentiator via a linkergroup (e.g. selected from, C₁-C₆alkylene, C₁-C₆alkenylene, arylene,heteroarylene, C₁-C₆alkyleneoxy and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂, etc.). Themodified compound can adsorb to an insoluble metal salt. Thus the methodmay further comprise: adsorbing the modified immunopotentiator to aninsoluble metal salt. This adsorption can modify or improve thepharmacokinetic profile of the modified immunopotentiator, and/or canincrease the time for which it is retained at a site of intramuscularinjection, relative to the unmodified immunopotentiator. The method mayfurther comprise a step of combining a modified adsorptiveimmunopotentiator with an immunogen e.g. to provide a vaccinecomposition.

According to a twenty-fourth aspect, the invention provides acomposition comprising (a) a compound according to formula (I) orformula (II), and (b) an immunogen.

According to a twenty-fifth-aspect, the invention provides a compositioncomprising: (a) an adjuvant complex comprising a TLR agonist adsorbed toan insoluble metal salt; and (b) at least two different immunogens. TheTLR agonist is preferably a TLR agonist as variously described abovee.g. in some embodiments the agonist is not a TLR4 agonist, in someembodiments the agonist is not a TLR9 agonist, in some embodiments it isof formula (I-A), etc. In some embodiments of this aspect, the TLRagonist is not compound 13 herein. In some embodiments of this aspect,the immunogen is not a three-protein mixture of 287-953, 936-741 and961c as disclosed in references 40 & 73. The invention also provides aprocess for preparing an immunogenic composition, comprising one of: (i)combining at least two different immunogens with a mixture comprising aTLR agonist and an insoluble metal salt; (ii) combining an insolublemetal salt with a mixture comprising a TLR agonist and at least twodifferent immunogens; (iii) combining a TLR agonist with a mixturecomprising an insoluble metal salt and at least two differentimmunogens; or (iv) combining in any order a TLR agonist, an insolublemetal salt, a first immunogen and a second immunogen.

According to a twenty-sixth aspect, the invention provides a compositioncomprising: (a) an adjuvant complex comprising a first TLR agonistadsorbed to an insoluble metal salt; and (b) an adjuvant complexcomprising a second TLR agonist adsorbed to an insoluble metal salt. Thecomposition can also include one or more immunogens.

According to a twenty-seventh aspect, the invention provides a processfor preparing an adjuvant complex, comprising steps of (i) preparing anaqueous mixture of a TLR agonist and a soluble aluminium salt; then (ii)adding a non-aluminium salt to the aqueous mixture in order to form aprecipitated aluminium salt to which the TLR agonist is adsorbed. TheTLR agonist is preferably a TLR agonist as variously described above.The invention also provides an adjuvant complex obtained or obtainableby this process. The complex can be mixed with an immunogen to providean immunogenic composition.

According to a twenty-eighth aspect, the invention provides a processfor preparing an immunogenic composition, comprising a step of mixing(i) an aqueous mixture of a TLR agonist and a soluble aluminium saltwith (ii) a buffered aqueous mixture of an immunogen, wherein the mixingstep causes precipitation of an aluminium salt to which the TLR agonistand the immunogen are adsorbed.

The TLR agonist is preferably a TLR agonist as variously describedabove. The invention also provides an immunogenic composition obtainedor obtainable by this process.

According to a twenty-ninth aspect, the invention provides an assay foranalysing an adjuvant complex which comprises a TLR agonist adsorbed toan insoluble metal salt, comprising steps of: (i) treating the complexto desorb TLR agonist from the insoluble metal salt; then (ii) detectingthe desorbed TLR agonist. Various other assays are also provided (seebelow).

In a thirtieth aspect, the invention provides a composition comprising aTLR agonist and an aluminium hydroxyphosphate adjuvant, wherein at least50% (e.g. ≧60%, ≧70%, ≧80%, ≧85%, ≧90%, ≧95%, ≧98%, ≧99%) of the TLRagonist is adsorbed onto the aluminium hydroxyphosphate, with theproviso that the TLR agonist:

-   -   (a) is not a TLR4 agonist; and    -   (b) is not a TLR9 agonist.

In some embodiments of this thirtieth aspect the TLR agonist is not acompound according to formula (III) as defined below and/or is not acompound of formula (I-A) as defined below.

According to a thirty-first aspect, the invention provides a method forpreparing an adjuvant complex, comprising steps of mixing an insolublealuminium salt with a complex of a TLR agonist and an insolublealuminium salt, thereby lowering the ratio of TLR agonist to aluminium.After mixing, the new mixture can be incubated to permit redistributionof the TLR agonist onto the added aluminium salt. This method permits abulk complex having a high SMIP:Al⁺⁺⁺ ratio to be diluted to a complexhaving a desired SMIP:Al⁺⁺⁺ ratio. The bulk can be used as the basis ofseveral dilutions, thus simplifying overall manufacture of multipledifferent end products. The insoluble aluminium salt which is added isideally free from TLR agonists, or else has a different SMIP:Al⁺⁺⁺ ratiothan the other material, thus permitting an overall change in ratio. Thealuminium salt in the two mixed materials is preferably the same salt.

Formulae (I) & (II)

In some embodiments of the invention, a TLR agonist of the invention isnot a compound according to formula (I) or (II). In other embodiments,however, TLR agonists of these formulae can be used e.g. in embodimentsaccording to the sixth aspect, where the TLR agonist is adsorbed to aninsoluble metal salt.

Formulae (I) and (II) correspond to the compounds disclosed in reference3. As used herein, formulae (I) and (II) are defined as follows:

wherein:

-   -   Z is —NH₂ or —OH;    -   X¹ is alkylene, substituted alkylene, alkenylene, substituted        alkenylene, alkynylene, substituted alkynylene, carbocyclylene,        substituted carbocyclylene, heterocyclylene, or substituted        heterocyclylene;    -   L¹ is a covalent bond, arylene, substituted arylene,        heterocyclylene, substituted heterocyclylene, carbocyclylene,        substituted carbocyclylene, —S—, —S(O)—, S(O)₂, —NR⁵—, or —O—    -   X² is a covalent bond, alkylene, or substituted alkylene;    -   L is NR⁵—, —N(R⁵)C(O)—, —O—, —S—, —S(O)—, S(O)₂, or a covalent        bond;    -   R³ is H, alkyl, substituted alkyl, heteroalkyl, substituted        heteroalkyl, alkenyl, substituted alkenyl, aryl, substituted        aryl, arylalkyl, substituted arylalkyl, heterocyclyl,        substituted heterocyclyl, heterocyclylalkyl, or substituted        heterocyclylalkyl;    -   Y¹ and Y² are each independently a covalent bond, —O— or —NR⁵—;        or —Y¹—R¹ and —Y²—R² are each independently —O—N═C(R⁶R⁷);

R¹ and R² are each independently H, alkyl, substituted alkyl,carbocyclyl, substituted carbocyclyl, heterocyclyl, substitutedheterocyclyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, arylalkyl, substituted arylalkyl, heterocyclylalkyl,substituted heterocyclylalkyl, -alkylene-C(O)—O—R⁵, -(substitutedalkylene)-C(O)—O—R⁵, -alkylene-O—C(O)—R⁵, -(substitutedalkylene)-O—C(O)—R⁵, -alkylene-O—C(O)—O—R⁵, or -(substitutedalkylene)-O—C(O)—O—R⁵

-   -   R⁴ is H, halogen, —OH, —O-alkyl, —O-alkylene-O—C(O)—O—R⁵,        —O—C(O)—O—R⁵, —SH, or —NH(R⁵);    -   each R⁵, R⁶, and R⁷ are independently H, alkyl, substituted        alkyl, carbocyclyl, substituted carbocyclyl, heterocyclyl,        substituted heterocyclyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, arylalkyl, substituted arylalkyl,        heterocyclylalkyl, or substituted heterocyclylalkyl.

Reference 3 discloses some compounds of formulae (I) and (II) whichinclude adsorptive moieties, but had not reported that these moietiescould be used for adsorption. Thus reference 3 does not realise thatthese moieties could be exploited to provide an adsorbed compound.

The invention also provides a composition comprising (a) a compound offormula (I) or formula (II) and (b) an insoluble metal salt; wherein thecompound of formula (I) or (II) is adsorbed onto the insoluble metalsalt.

Formula (III)

In some embodiments of the invention, a TLR agonist of the invention isnot a compound according to formula (III). In other embodiments,however, TLR agonists of formula (III) can be used e.g. in embodimentsaccording to the twentieth aspect, where the TLR agonist is adsorbed toan insoluble metal salt and then sterilised.

Formula (III) corresponds to compounds disclosed in reference 9. As usedherein, formula (III) is defined as follows:

wherein:

-   -   R¹ is H, —C(O)—C₁₀-C₁₈alkyl; R² is C₁₀-C₁₈alkyl; R³ is        C₁₀-C₁₈alkyl;

L₁ is —CH₂O—, —CH₂OC(O)—, —CH₂NR⁷C(O)— or —C(O)NR⁷—;

L₂ is —O—, —OC(O)— or —NR⁷C(O)—;

-   -   R⁴ is -L₃R⁵ or -L₄R⁵;    -   R⁵ is —N(R⁷)₂, —OR⁷, —P(O)(OR⁷)₂, —C(O)OR⁷, —NR⁷C(O)L₃R⁸,        —OL₃R⁶—, —C(O)NR⁷L₃R⁸, C₁-C₆alkyl, a C₆ aryl, a C₁₀ aryl, a C₁₄        aryl, 5 to 14 membered heteroaryl containing 1 to 3 heteroatoms        selected from O, S and N, C₃-C₈cycloalkyl or a 5 to 6 membered        heterocycloalkyl containing 1 to 3 heteroatoms selected from O,        S and N, wherein the aryl, heteroaryl, cycloalkyl and        heterocycloalkyl of R⁵ are each optionally substituted with 1 to        3 substituents independently selected from —OR⁹, —OL₃R⁶, —OL₄R⁶,        —OR⁷, and —C(O)OR⁷;    -   L₃ is a C₁-C₁₀alkylene, wherein the C₁-C₆alkylene of L₃ is        optionally substituted with 1 to 4 R⁶ groups, or the        C₁-C₆alkylene of L₃ is substituted with 2 C₁-C₆alkyl groups on        the same carbon atom which together, along with the carbon atom        they are attached to, form a C₃-C₈cycloakyl;    -   L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)— or        —(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—, wherein each R¹¹ is        a C₁-C₆alkyl groups which together, along with the carbon atom        they are attached to, form a C₃-C₈cycloakyl;    -   each R⁶ is independently selected from halo, C₁-C₆alkyl, —OR⁷,        —N(R⁷)₂, —C(O)N(R⁷)₂, —P(O)(OR⁷)₂, a C₆ aryl, a C₁₀ aryl and a        C₁₄ aryl;    -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is selected from —SR⁷, —C(O)OH and a 5 to 6 membered        heterocycloalkyl containing 1 to 3 heteroatoms selected from O        and N;    -   R⁹ is phenyl;    -   each R¹⁰ is independently selected from H and halo;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and    -   q is 1, 2, 3 or 4.

In some embodiments of the invention, a TLR agonist of the invention isa compound which is according to formula (IIIa) but which is notaccording to formula (III):

wherein:

-   -   R¹ is H, —C(O)—C₇-C₁₈alkyl or —C(O)—C₁-C₆alkyl;    -   R² is C₇-C₁₈alkyl;    -   R³ is C₇-C₁₈alkyl;    -   L₁ is —CH₂OC(O)—, —CH₂O—, —CH₂NR⁷C(O)— or —CH₂OC(O)NR⁷—;    -   L₂ is —OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—;    -   R⁴ is -L₃R⁵ or -L₄R⁵;    -   R⁵ is —N(R⁷)₂, —OR⁷, —P(O)(OR⁷)₂, —C(O)OR⁷, —NR⁷C(O)L₃R⁸,        —NR⁷C(O)L₄R⁸, —OL₃R⁶, —C(O)NR⁷L₃R⁸, —C(O)NR⁷L₄R⁸, —S(O)₂OR⁷,        —OS(O)₂OR⁷, C₁-C₆alkyl, a C₆aryl, a C₁₀aryl, a C₁₄aryl, 5 to 14        ring membered heteroaryl containing 1 to 3 heteroatoms selected        from O, S and N, C₃-C₈cycloalkyl or a 5 to 6 ring membered        heterocycloalkyl containing 1 to 3 heteroatoms selected from O,        S and N, wherein the aryl, heteroaryl, cycloalkyl and        heterocycloalkyl of R⁵ are each unsubstituted or the aryl,        heteroaryl, cycloalkyl and heterocycloalkyl of R⁵ are each        substituted with 1 to 3 substituents independently selected from        —OR⁹, —OL₃R⁶, —OL₄R⁶, —OR⁷, and —C(O)OR⁷;    -   L₃ is a C₁-C₁₀alkylene, wherein the C₁-C₁₀alkylene of L₃ is        unsubstituted, or the C₁-C₁₀alkylene of L₃ is substituted with 1        to 4 R⁶ groups, or the C₁-C₁₀alkylene of L₃ is substituted with        2 C₁-C₆alkyl groups on the same carbon atom which together,        along with the carbon atom they are attached to, form a        C₃-C₈cycloakyl;    -   L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)— or        —(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—, wherein each R¹¹ is        a C₁-C₆alkyl groups which together, along with the carbon atom        they are attached to, form a C₃-C₈cycloakyl;    -   each R⁶ is independently selected from halo, C₁-C₆alkyl,        C₁-C₆alkyl substituted with 1-2 hydroxyl groups, —OR⁷, —N(R⁷)₂,        —C(O)OH, —C(O)N(R⁷)₂, —P(O)(OR⁷)₂, a C₆aryl, a C₁₀aryl and a        C₁₄aryl;    -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   R⁸ is selected from —SR⁷, —C(O)OH, —P(O)(OR⁷)₂, and a 5 to 6        ring membered heterocycloalkyl containing 1 to 3 heteroatoms        selected from O and N;    -   R⁹ is phenyl;    -   each R¹⁰ is independently selected from H and halo;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and    -   q is 1, 2, 3 or 4.

These compounds which fall inside formula (IIIa), but outside formula(III), may overlap with formula (F) and/or formula (J).

Even where compounds of formula (III) are disclaimed, in someembodiments the invention may nevertheless use one of the followingcompounds 72 to 101:

These 30 compounds (compounds 72 to 101, collectively the “TLR2p”compounds), which are generally of use with the invention, can be madeusing the protocols described in reference 9. An exemplary synthesis isincluded below.

Formula (I-A)

In some embodiments of the invention, a TLR agonist of the invention isnot a compound according to formula (I-A). In other embodiments,however, TLR agonists of formula (I-A) can be used e.g. in embodimentsaccording to the twentieth aspect, where the TLR agonist is adsorbed toan insoluble metal salt and then sterilised.

Formula (I-A) corresponds to the compounds disclosed in reference 4. Asused herein, formula (I-A) is defined as follows:

-   -   wherein:        -   X³ is N;        -   X⁴ is N or CR³        -   X⁵ is —CR⁴═CR⁵—;        -   R¹ and R² are H;        -   R³ is H;        -   R⁴ and R⁵ are each independently selected from H, halogen,            —C(O)OR⁷, —C(O)R⁷, —C(O)N(R¹¹R¹²), —N(R¹¹R¹²), —N(R⁹)₂,            —NHN(R⁹)₂, —SR⁷, —(CH₂)_(n)OR⁷, —(CH₂)_(n)R⁷, -LR⁸, - LR¹⁰,            —OLR⁸, —OLR¹⁰, C₁-C₆alkyl, C₁-C₆hetcroalkyl, C₁-C₆haloalkyl,            C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy,            aryl, heteroaryl, C₃-C₈cycloalkyl, and            C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl,            C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne,            C₁-C₆alkoxy, C₁-C₆haloalkoxy, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl groups of R⁴ and            R⁵ are each optionally substituted with 1 to 3 substituents            independently selected from halogen, —CN, —NO₂, —R⁷, —OR⁸,            —C(O)R⁸, —OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂,            —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂, —C(O)N(R⁹)₂,            —S(O)₂R⁸, —S(O)R⁸, —S(O)₂N(R⁹)₂, and —NR⁹S(O)₂R⁸;        -   or, R³ and R⁴, or R⁴ and R⁵, or R⁵ and R⁶, when present on            adjacent ring atoms, can optionally be linked together to            form a 5-6 membered ring, wherein the 5-6 membered ring is            optionally substituted with R⁷;        -   each L is independently selected from a bond,            —(O(CH₂)_(m))_(t)—, C₁-C₆alkyl, C₂-C₆alkenylene and            C₂-C₆alkynylene, wherein the C₁-C₆alkyl, C₂-C₆alkenylene and            C₂-C₆alkynylene of L are each optionally substituted with 1            to 4 substituents independently selected from halogen, —R⁸,            —OR⁸, —N(R⁹)₂, —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, and            —OP(O)(OR¹⁰)₂;        -   R⁷ is selected from H, C₁-C₆alkyl, aryl, heteroaryl,            C₃-C₈cycloalkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl,            C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy, C₁-C₆haloalkoxy, and            C₃-C₈heterocycloalkyl, wherein the C₁-C₆alkyl, aryl,            heteroaryl, C₃-C₈cycloalkyl, C₁-C₆heteroalkyl,            C₁-C₆haloalkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆alkoxy,            C₁-C₆haloalkoxy, and C₃-C₈heterocycloalkyl groups of R⁷ are            each optionally substituted with 1 to 3 R¹³ groups, and each            R¹³ is independently selected from halogen, —CN, -LR⁹,            -LOR⁹, —OLR⁹, -LR¹⁰, -LOR¹⁰, —OLR¹⁰, -LR⁸, -LOR⁸, —OLR⁸,            -LSR⁸, -LSR¹⁰, -LC(O)R⁸, —OLC(O)R₈, -LC(O)OR⁸, -LC(O)R¹⁰,            -LOC(O)OR⁸, -LC(O)NR⁹R¹¹, -LC(O)NR⁹R⁸, -LN(R⁹)₂, -LNR⁹R⁸,            -LNR⁹R¹⁰, -LC(O)N(R⁹)₂, -LS(O)₂R⁸, -LS(O)R⁸, -LC(O)NR⁸OH,            -LNR⁹C(O)R⁸, -LNR⁹C(O)OR⁸, -LS(O)₂N(R⁹)₂, —OLS(O)₂N(R⁹)₂,            -LNR⁹S(O)₂R⁸, -LC(O)NR⁹LN(R⁹)₂, -LP(O)(OR⁸)₂, -LOP(O)(OR⁸)₂,            -LP(O)(OR¹⁰)₂ and —OLP(O)(OR¹⁰)₂;        -   each R⁸ is independently selected from H, —CH(R¹⁰)₂,            C₁-C₈alkyl, C₂-C₈alkene, C₂-C₈alkyne, C₁-C₆haloalkyl,            C₁-C₆alkoxy, C₁-C₆heteroalkyl, C₃-C₈cycloalkyl,            C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl and            C₁-C₆haloalkoxy, wherein the C₁-C₈alkyl, C₂-C₈alkene,            C₂-C₈alkyne, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, C₁-C₆alkoxy,            C₃-C₈cycloalkyl, C₂-C₈heterocycloalkyl, C₁-C₆hydroxyalkyl            and C₁-C₆haloalkoxy groups of R⁸ are each optionally            substituted with 1 to 3 substituents independently selected            from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, —OC(O)R¹¹,            —C(O)N(R⁹)₂, —C(O)OR¹¹, —NR⁹C(O)R¹¹, —NR⁹R¹⁰, —NR¹¹R¹²,            —N(R⁹)₂, —OR⁹, —OR¹⁰, —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹,            —S(O)R¹¹, —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂, and            —OP(O)(OR¹¹)₂;        -   each R⁹ is independently selected from H, —C(O)R⁸, —C(O)OR⁸,            —C(O)R¹⁰, —C(O)OR¹⁰, —S(O)₂R¹⁰, —C₁-C₆ alkyl, C₁-C₆            heteroalkyl and C₃-C₆ cycloalkyl, or each R⁹ is            independently a C₁-C₆alkyl that together with N they are            attached to form a C₃-C₈heterocycloalkyl, wherein the            C₃-C₈heterocycloalkyl ring optionally contains an additional            heteroatom selected from N, O and S, and wherein the C₁-C₆            alkyl, C₁-C₆ heteroalkyl, C₃-C₆ cycloalkyl, or            C₃-C₈heterocycloalkyl groups of R⁹ are each optionally            substituted with 1 to 3 substituents independently selected            from —CN, R¹¹, —OR¹¹, —SR¹¹, —C(O)R¹¹, OC(O)R¹¹, —C(O)OR¹¹,            —NR¹¹R¹², —C(O)NR¹¹R¹², —C(O)NR¹¹OH, —S(O)₂R¹¹, —S(O)R¹¹,            —S(O)₂NR¹¹R¹², —NR¹¹S(O)₂R¹¹, —P(O)(OR¹¹)₂ and            —OP(O)(OR¹¹)₂;        -   each R¹⁰ is independently selected from aryl,            C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and heteroaryl,            wherein the aryl, C₃-C₈cycloalkyl, C₃-C₈heterocycloalkyl and            heteroaryl groups are optionally substituted with 1 to 3            substituents selected from halogen, —R⁸, —OR⁸, -LR⁹, -LOR⁹,            —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹CO₂R⁸. —CO₂R⁸, —C(O)R⁸ and            —C(O)N(R⁹)₂;        -   R¹¹ and R¹² are independently selected from H, C₁-C₆alkyl,            C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl, heteroaryl,            C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl, wherein the            C₁-C₆alkyl, C₁-C₆heteroalkyl, C₁-C₆haloalkyl, aryl,            heteroaryl, C₃-C₈cycloalkyl, and C₃-C₈heterocycloalkyl            groups of R¹¹ and R¹² are each optionally substituted with 1            to 3 substituents independently selected from halogen, —CN,            R⁸, —OR⁸, C(O)R₈, OC(O)R⁸, —C(O)OR⁸, —N(R⁹)₂, —NR⁸C(O)R⁸,            —NR⁸C(O)OR⁸, —C(O)N(R⁹)₂, C₃-C₈heterocycloalkyl, —S(O)₂R⁸,            —S(O)₂N(R⁹)₂, —NR⁹S(O)₂R⁸, C₁-C₆haloalkyl and            C₁-C₆haloalkoxy;        -   or R¹¹ and R¹² are each independently C₁-C₆alkyl and taken            together with the N atom to which they are attached form an            optionally substituted C₃-C₈heterocycloalkyl ring optionally            containing an additional heteroatom selected from N, O and            S;        -   ring A is an aryl or a heteroaryl, wherein the aryl and            heteroaryl groups of Ring A are optionally substituted with            1 to 3 R^(A) groups, wherein each R^(A) is independently            selected from —R⁸, —R⁷, —OR⁷, —OR⁸, —R¹⁰, —OR¹⁰, —SR⁸, —NO₂,            —CN, —N(R⁹)₂, —NR⁹C(O)R⁸, —NR⁹C(S)R⁸, —NR⁹C(O)N(R⁹)₂,            —NR⁹C(S)N(R⁹)₂, —NR⁹CO₂R⁸, —NR⁹NR⁹C(O)R⁸, —NR⁹NR⁹C(O)N(R⁹),            —NR⁹NR⁹CO₂R⁸, —C(O)C(O)R⁸, —C(O)CH₂C(O)R⁸, —CO₂R,            —(CH₂)_(n)CO₂R⁸, —C(O)R⁸, —C(S)R⁸, —C(O)N(R⁹)₂, —C(S)N(R⁹)₂,            —OC(O)N(R⁹)₂. —OC(O)R⁸, —C(O)N(OR⁸)R⁸, —C(NOR⁸)R⁸, —S(O)₂R⁸,            —S(O)₃R⁸, —SO₂N(R⁹)₂, —S(O)R⁸, —NR⁹SO₂N(R⁹)₂, —NR⁹SO₂R⁸,            —P(O)(OR⁸)₂, —OP(O)(OR⁸)₂, —P(O)(OR¹⁰)₂, —OP(O)(OR¹⁰)₂,            —N(OR⁸)R⁸, —CH═CHCO₂R⁸, —C(═NH)—N(R⁹)₂, and            —(CH₂)_(n)NHC(O)R⁸ or two adjacent R^(A) substituents on            Ring A form a 5-6 membered ring that contains up to two            heteroatoms as ring members;        -   n is, independently at each occurrence, 0, 1, 2, 3, 4, 5, 6,            7 or 8;        -   each m is independently selected from 1, 2, 3, 4, 5 and 6,            and        -   t is 1, 2, 3, 4, 5, 6, 7 or 8.

Reference 4 discloses compounds of formula (I-A) which have intrinsicadsorptive properties, but the inventors did not report at the time offiling reference 4 that these properties exist and thus did not reportthat they could be exploited to provide an adsorbed compound.

Formula (IV)

In some embodiments of the invention, a TLR agonist of the invention isnot a compound according to formula (IV). Formula (IV) corresponds tocompounds disclosed in reference 10. As used herein, formula (IV) isdefined as follows:

wherein:

-   -   R¹ is H, C₁-C₆alkyl, —C(R⁵)₂OH, -L¹R⁵, -L¹R⁶, -L²R⁵, -L²R⁶,        —OL²R⁵, or —OL²R⁶;    -   L¹ is —C(O)— or —O—;    -   L² is C₁-C₆alkylene, C₂-C₆alkenylene, arylene, heteroarylene or        —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p)—, wherein the C₁-C₆alkylene and        C₂-C₆alkenylene of L² are optionally substituted with 1 to 4        fluoro groups;    -   each L³ is independently selected from C₁-C₆alkylene and        —((CR⁴R⁴)_(p)O)_(q)(CH₂)_(p)—, wherein the C₁-C₆alkylene of L³        is optionally substituted with 1 to 4 fluoro groups;    -   L⁴ is arylene or heteroarylene;    -   R² is H or C₁-C₆alkyl;    -   R³ is selected from C₁-C₄alkyl, -L³R⁵, -L¹R⁵, -L³R⁷, -L³L⁴L³R⁷,        -L³L⁴R⁵, -L³L⁴L³R⁵, —OL³R⁵, —OL³R⁷, —OL³L⁴R⁷, —OL³L⁴L³R⁷, —OR⁸,        —OL³L⁴R⁵, —OL³L⁴L³R⁵ and —C(R⁵)₂OH;    -   each R⁴ is independently selected from H and fluoro;    -   R⁵ is —P(O)(OR⁹)₂,    -   R⁶ is —CF₂P(O)(OR⁹)₂ or —C(O)OR¹⁰;    -   R⁷ is —CF₂P(O)(OR⁹ or —C(O)OR¹⁰;    -   R⁸ is H or C₁-C₄alkyl;    -   each R⁹ is independently selected from H and C₁-C₆alkyl;

R¹⁰ is H or C₁-C₄alkyl;

-   -   each p is independently selected from 1, 2, 3, 4, 5 and 6, and    -   q is 1, 2, 3 or 4.

Formulae (C), (D), (E), (G) and (H)

As discussed above, the sixteenth aspect of the invention provides TLR7agonists of formulae (C), (D), (E), or (H). The sixteenth aspect of theinvention provides compounds of formula (C). The sixteenth aspect of theinvention also and independently provides compounds of formula (D). Thesixteenth aspect of the invention also and independently providescompounds of formula (E). The sixteenth aspect of the invention also andindependently provides compounds of formula (H).

The ‘parent’ compounds of formulae (C), (D), (E) and (H) are useful TLR7agonists (see references 2-5 and 11-27) but are modified herein byattachment of a phosphorus-containing moiety. Compounds of formulae (C),(D), (E) and (H) can thus be used with various aspects of the invention.

In some embodiments of formulae (C), (D) and (E) the compounds havestructures according to formulae (C′), (D′) and (E′), shown below:

The embodiments of the invention of formulae (C), (D), (E) and (H) alsoapply to formulae (C′), (D′), (E′) and (H′).

In some embodiments of formulae (C), (D), (E), and (H): X is O; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (C): P³ is selected from C₁-C₆alkyl,CF₃, and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and—Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴ is selected from —C₁-C₆alkylaryl and—Y-L-X—P(O)(OR^(X))(OR^(Y)); X^(C) is CH; X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; q is 1 or 2.

In other embodiments of formulae (C), (D), (E), and (H): X is a covalentbond; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)—each optionally substituted with 1 to 4 substituents independentlyselected from halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each pis independently selected from 1, 2 and 3; and q is selected from 1 and2.

In other embodiments of formula (C): P is selected from C₁-C₆alkyl, CF₃,and —((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁴is selected from —C₁-C₆alkylaryl and —Y-L-X—P(O)(OR^(X))(OR^(Y)); X^(C)is N; X is a covalent bond; L is selected from C₁-C₆alkylene and—((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4substituents independently selected from halo, OH, C₁-C₄alkyl,—OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2and 3; q is selected from 1 and 2.

In other embodiments of formula (D): P⁵ is selected from C₁-C₆alkyl, and—Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (D): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (D): X is a covalent bond; L is selectedfrom C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X is a covalent bond; L is selectedfrom C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In other embodiments of formula (E): X^(E) is CH₂, P⁸ is C₁-C₆alkoxyoptionally substituted with —Y-L-X—P(O)(OR^(X))(OR^(Y)).

In other embodiments of formula (E): P⁹ is —NHC₁-C₆alkyl optionallysubstituted with OH and C₁-C₆alkyl, and —Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, a compound of formula (C) is not a compound inwhich P⁴ is —Y-L-X—P(O)(OR^(X))(OR^(Y)).

In some embodiments, in a compound of formula (C), P is selected from H,C₁-C₆alkyl, —C₁-C₆alkylaryl.

Preferred compounds of formulae (C), (D) and (E) are compounds (6), (7),(8), (67), (68), (69) and (70) as disclosed below. The inventionprovides each of compounds (6), (7), (8), (67), (68), (69) and (70).

In some embodiments of formula (H): X^(H1)-X^(H2) is CR^(H2)R^(H3),R^(H2) and R^(H3) are H, X^(H3) is N, X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments of formula (H): X^(H1)-X^(H2) is CR^(H2)R^(H3),R^(H2) and R^(H3) are H, X^(H3) is N, X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments, a TLR agonist of the invention, for example, acompound of formula (H), is not one of the following two compounds;

As discussed above, the seventeenth aspect of the invention providescompounds of formula (G).

The ‘parent’ compounds of formula (G) are useful TLR8 agonists (seereferences 6 & 7) but are modified herein by attachment of aphosphorus-containing moiety. Compounds of formulae (G) can thus be usedwith various aspects of the invention.

In some embodiments of formula (G), the compounds have structuresaccording to formula (G′);

In some embodiments of formula (G) or (G′): X^(G) is C and

represents a double bond.

In some embodiments of formula (G) or (G′): X is a covalent bond; L isselected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

In some embodiments of formula (G) or (G′): X is O; L is selected fromC₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; each p is independentlyselected from 1, 2 and 3; and q is selected from 1 and 2.

Formula (B)

The eighteenth aspect of the invention provides a compound according toformula (B)

wherein:

-   -   P¹ is selected from H, C₁-C₆alkyl optionally substituted with        COOH and —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   P² is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy and        —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   with the proviso that at least one of P¹ and P² is        —Y-L-X—P(O)(OR^(X))(OR^(Y));    -   R^(B) is selected from H and C₁-C₆alkyl;    -   R^(X) and R^(Y) are independently selected from H and        C₁-C₆alkyl;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6; and    -   q is selected from 1, 2, 3 and 4.

In some embodiments of formula (B): P¹ is selected from C₁-C₆alkyloptionally substituted with COOH and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P² isselected from C₁-C₆alkoxy and —Y-L-X—P(O)(OR^(X))(OR^(Y)); R^(B) isC₁-C₆alkyl; X is a covalent bond; L is selected from C₁-C₆alkylene and—((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4substituents independently selected from halo, OH, C₁-C₄alkyl,—OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2and 3; q is selected from 1 and 2.

In some embodiments of the invention (e.g. those where the compound offormula (B) is adsorbed to an aluminium salt), a TLR agonist of theinvention is not one of the following four compounds:

In some embodiments of the invention (e.g. the 25th and 26th aspects)preferred compounds of formula (B) are compounds 1, 2, 5, and 13 herein.

Formula (F)

The eighteenth aspect of the invention provides a compound according toformula (F):

wherein:

-   -   R^(X) and R^(Y) are independently selected from H and        C₁-C₆alkyl;    -   Q and V are independently selected from a covalent bond, NH, S,        C(O) and O;    -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is selected from, a covalent bond C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; and    -   R^(F1), R^(F2) and R^(F3) are independently selected from H,        C₁-C₁₈ alkyl, C₁-C₁₈alkenyl, C(O)C₁-C₁₈alkyl and        C(O)C₁-C₁₈alkenyl;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6; and    -   q is selected from 1, 2, 3 and 4.

The ‘parent’ compounds of formula (F) are useful TLR2 agonists (seereference 9) but are modified herein by attachment of aphosphorus-containing moiety. Compounds of formulae (F) can thus be usedwith various aspects of the invention.

In some embodiments, compounds of formula (F) are not compoundsaccording to formula (III), as defined above.

In some embodiments, a TLR agonist of the invention, for example, acompound of formula (F), is not one of the following seven compounds(16) to (22):

In some embodiments (e.g. those where the compound of formula (F) isadsorbed to an aluminium salt), a TLR agonist of the invention is notone of compounds (17), (19) or (22).

In some embodiments of formula (F): Q and V are O.

In some embodiments of formula (F): X is O.

In some embodiments of formula (F): Y is O.

In some embodiments of formula (F): R^(X) and R^(Y) are H; Q and V areO; X is selected from a covalent bond and O; Y is NH; L is selected fromC₁-C₆alkylene, arylene, heteroarylene, —((CH₂)_(p)O)_(q)(CH₂)_(p)— eachoptionally substituted with 1 to 4 substituents independently selectedfrom halo, OH, C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂; R^(F1), R^(F2)and R^(F3) are selected from C₁-C₁₈ alkyl and C(O)C₁-C₁₈alkyl; each p isindependently selected from 1, 2 and 3; and q is selected from 1 and 2.

Formula (J)

The eighteenth aspect of the invention provides a compound according toformula (J):

wherein:

-   -   R¹ is H, —C(O)—C₇-C₁₈alkyl or —C(O)—C₁-C₆alkyl;    -   R² is C₇-C₁₈alkyl;    -   R³ is C₁-C₁₈alkyl;    -   L₁ is —CH₂OC(O)—, —CH₂O—, —CH₂NR⁷C(O)— or —CH₂OC(O)NR⁷—;    -   L₂ is —OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—;    -   R⁴ is -L₃R⁵ or -L₄R⁵;    -   R⁵ is P(O)(OR⁷)₂, —NR⁷C(O)L₃-P(O)(OR⁷)₂, —NR⁷C(O)L₄-P(O)(OR)₂,        —OL₃-P(O)(OR⁷)₂, —C(O)NR⁷L₃-P(O)(OR⁷)₂, or        —C(O)NR⁷L₄-P(O)(OR⁷)₂,    -   L₃ is a C₁-C₁₀alkylene, wherein the C₁-C₁₀alkylene of L₃ is        unsubstituted, or the C₁-C₁₀alkylene of L₃ is substituted with 1        to 4 R⁶ groups, or the C₁-C₁₀alkylene of L₃ is substituted with        2 C₁-C₆alkyl groups on the same carbon atom which together,        along with the carbon atom they are attached to, form a        C₃-C₈cycloakyl;    -   L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)— or        —(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰OR¹⁰)_(p)—, wherein each R¹¹ is        a C₁-C₆alkyl groups which together, along with the carbon atom        they are attached to, form a C₃-C₈cycloakyl;    -   each R⁶ is independently selected from halo, C₁-C₆alkyl,        C₁-C₆alkyl substituted with 1-2 hydroxyl groups, —OR⁷, —N(R⁷)₂,        —C(O)OH, —C(O)N(R⁷)₂, —P(O)(OR⁷)₂, a C₆aryl, a C₁₀aryl and a        C₁₄aryl;    -   each R⁷ is independently selected from H and C₁-C₆alkyl;    -   each R¹⁰ is independently selected from H and halo;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6; and    -   q is 1, 2, 3 or 4.

In some embodiments of (J), R₁ is H. In other embodiments of (J), R₁ is—C(O)—C₁₅alkyl;

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—, —O—,—NR⁷C(O)— or —OC(O)NR⁷—; or (ii) or L₁ is —CH₂O— and L₂ is —OC(O)—, —O—,—NR⁷C(O)— or —OC(O)NR⁷—; or (iii) L₁ is —CH₂NR⁷C(O)— and L₂ is —OC(O)—,—O—, —NR⁷C(O)— or —OC(O)NR⁷—; or (iv) L is —CH₂OC(O)NR⁷— and L₂ is—OC(O)—, —O—, —NR⁷C(O)— or —OC(O)NR⁷—.

In some embodiments of (J): (i) L₁ is —CH₂OC(O)— and L₂ is —OC(O)—; or(ii) L₁ is —CH₂O— and L₂ is —O—; or (iii) L₁ is —CH₂O— and L₂ is—NHC(O)—; or (iv) L₁ is —CH₂OC(O)NH— and L is —OC(O)NH—.

In some embodiments of (J), (i) R² is —C₁₁alkyl and R³ is —C₁₁alkyl; or(ii) R² is —C₁₆alkyl and R³ is —C₁₆alkyl; or (iii) R² is —C₁₆alkyl andR³ is —C₁₁alkyl; or (iv) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (v) R²is —C₇alkyl and R³ is —C₇alkyl; or (vi) R² is —C₉alkyl and R³ is—C₉alkyl; or (vii) R² is —C₈alkyl and R³ is —C₈alkyl; or (viii) R² is—C₁₃alkyl and R³ is —C₁₃alkyl; or (ix) R² is —C₁₂alkyl and R³ is—C₁₁alkyl; or (x) R² is —C₁₂alkyl and R³ is —C₁₂alkyl; or (xi) R² is—C₁₀alkyl and R³ is —C₁₀alkyl; or (xii) R² is —C₁₅alkyl and R³ is—C₁₅alkyl.

In some embodiments of (J), R² is —C₁₁alkyl and R³ is —C₁₁alkyl.

In some embodiments of (J), L₃ is a C₁-C₁₀alkylene, wherein theC₁-C₁₀alkylene of L₃ is unsubstituted or is substituted with 1 to 4 R⁶groups.

In some embodiments of (J): L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—;each R¹⁰ is independently selected from H and F; and each p isindependently selected from 2, 3, and 4.

In some embodiments of (J), each R⁶ is independently selected frommethyl, ethyl, i-propyl, i-butyl, —CH₂OH, —OH, —F, —NH₂, —C(O)OH,—C(O)NH₂, —P(O)(OH)₂ and phenyl.

In some embodiments of (J), each R⁷ is independently selected from H,methyl and ethyl.

Specific compounds of formula (J) which are useful with the inventioninclude the “TLR2p” group disclosed above. Further specific compounds offormula (J) which are useful include:

-   (3-((R)-2-amino-3-(((R)-2,3-bis(dodecanoyloxy)propyl)thio)propanamido)propyl)phosphonic    acid; (16)-   ((8R,12R)-8-amino-12-(dodecanoyloxy)-7,15-dioxo-3,14-dioxa-10-thia-6-azahexacosyl)phosphonic    acid; (17)-   ((12R,16R)-12-amino-16-(dodecanoyloxy)-1,1-difluoro-11,19-dioxo-4,7,18-trioxa-14-thia-10-azatriacontyl)phosphonic    acid; (18)-   ((11R,15R)-1-amino-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-13-thia-9-azanonacosyl)phosphonic    acid; (19)-   ((6S,9R,13R)-9-amino-13-(dodecanoyloxy)-6-methyl-8,16-dioxo-4,15-dioxa-11-thia-7-azaheptacosyl)phosphonic    acid; (21)-   (3-((1-((R)-2-amino-3-(((R)-2,3-bis(dodecanoyloxy)propyl)thio)propanamido)cyclopropyl)methoxy)propyl)phosphonic    acid; (22)-   (3-(4-(2-((R)-2-amino-3-(((R)-2,3-bis(dodecanoyloxy)propyl)thio)propanamnido)ethyl)phenoxy)propyl)phosphonic    acid; (20)-   ((14R,18R)-14-amino-18-(dodecanoyloxy)-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12-azadotriacontyl)phosphonic    acid; (72)-   (4-((R)-2-amino-3-(((R)-2,3-bis(dodecanoytoxy)propyl)thio)propanamido)-1,1-difluorobutyl)phosphonic    acid; (73)-   ((14R,15R)-14-amino-18-(dodecyloxy)-13-oxo-3,6,9,20-tetraoxa-16-thia-12-azadotriacontyl)phosphonic    acid; (74)-   ((9R,13R)-9-amino-13-(dodecanoyloxy)-1,1-difluoro-8,16-dioxo-4,15-dioxa-11-thia-7-azaheptacosyl)phosphonic    acid; (75)-   ((12R,16R)-12-amino-16-(dodecyloxy)-1,1-difluoro-11-oxo-4,7,18-trioxa-14-thia-10-azatriacontyl)phosphonic    acid; (76)-   ((14R,18R)-14-amino-18-(octanoyloxy)-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12-azaoctacosyl)phosphonic    acid; (77)-   ((14R,18R)-14-amino-18-(decanoyloxy)-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12-azatriacontyl)phosphonic    acid; (78)-   ((14R,18R)-14-amino-13,21-dioxo-18-(tetradecanoyloxy)-3,6,9,20-tetraoxa-16-thia-12-azatetratriacontyl)phosphonic    acid; (79)-   ((14R,18R)-14-amino-18-dodecanamido-13-oxo-3,6,9,20-tetraoxa-16-thia-12-azadotriacontyl)phosphonic    acid; (80)-   ((14R,18R)-14-amino-18-(dodecanoyloxy)-13-oxo-3,6,9,20-tetraoxa-16-thia-12-azadotriacontyl)phosphonic    acid; (81)-   ((11S,14R,18R)-14-amino-18-dodecanamido-11-methyl-13-oxo-3,6,9,20-tetraoxa-16-thia-12-azadotriacontyl)phosphonic    acid; (82)-   ((11S,14R,18R)-14-amino-18-(dodecyloxy)-11-methyl-13-oxo-3,6,9,20-tetraoxa-16-thia-12-azadotriacontyl)phosphonic    acid; (83)-   ((11S,14R,18R)-14-amino-18-(dodecyloxy)-11-methyl-10,13-dioxo-3,6,20-trioxa-16-thia-9,12-diazadotriacontyl)phosphonic    acid; (84)-   ((11S,14R,18R)-14-amino-18-dodecanamido-11-methyl-10,13-dioxo-3,6,20-trioxa-16-thia-9,12-diazadotriacontyl)phosphonic    acid; (85)-   ((14R,18R)-18-(dodecanoyloxy)-13,21-dioxo-14-palmitamido-3,6,9,20-tetraoxa-16-thia-12-azadotriacontyl)phosphonic    acid; (86)-   ((12R,16R)-12-amino-16-dodecanamido-1,1-difluoro-11-oxo-4,7,18-trioxa-14-thia-10-azatriacontyl)phosphonic    acid; (87)-   ((14R,18R)-14-amino-18-((decylcarbamoyl)oxy)-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12,22-diazadotriacontyl)phosphonic    acid; (88)-   ((14R,18R)-14-amino-18-((octylcarbamoyl)oxy)-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12,22-diazatriacontyl)phosphonic    acid; (89)-   ((14R,18R)-18-((decylcarbamoyl)oxy)-13,21-dioxo-14-palmitamido-3,6,9,20-tetraoxa-16-thia-12,22-diazadotriacontyl)phosphonic    acid; (90)-   ((14R,18R)-14-amino-18-(hexadecyloxy)-13-oxo-3,6,9,20-tetraoxa-16-thia-12-azahexatriacontyl)phosphonic    acid; (91)-   ((17R,21R)-7-amino-21-(dodecanoyloxy)-16,24-dioxo-3,6,9,12,23-pentaoxa-19-thia-15-azapentatriacontyl)phosphonic    acid; (92)-   ((17R,21R)-17-amino-21-(dodecyloxy)-16-oxo-3,6,9,12,23-pentaoxa-19-thia-15-azapentatriacontyl)phosphonic    acid; (93)-   ((17R,21R)-17-amino-21-dodecanamido-16-oxo-3,6,9,12,23-pentaoxa-19-thia-15-azapentatriacontyl)phosphonic    acid; (94)-   ((11S,14R,18R)-14-amino-18-(dodecyloxy)-11-(hydroxymethyl)-10,13-dioxo-3,6,20-trioxa-16-thia-9,12-diazadotriacontyl)phosphonic    acid; (95)-   ((11S,14R,18R)-14-amino-18-dodecanamido-1-(hydroxymethyl)-10,13-dioxo-3,6,20-trioxa-16-thia-9,12-diazadotriacontyl)phosphonic    acid; (96)-   ((14R,18R)-14-acetamido-18-((decylcarbamoyl)oxy)-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12,22-diazadotriacontyl)phosphonic    acid; (97)-   ((14R,18R)-14-amino-18-((decylcarbamoyl)oxy)-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12,22-diazadotriacontyl)phosphonic    acid; (98)-   ((14R,18R)-18-((decylcarbamoyl)oxy)-14-heptanamido-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12,22-diazadotriacontyl)phosphonic    acid; (99)-   ((14R,18R)-14-amino-18-((dodecyloxy)methyl)-13,20-dioxo-3,6,9,19-tetraoxa-16-thia-12,21-diazahentriacontyl)phosphonic    acid; (100)-   ((14R,18R)-18-((decylcarbamoyl)oxy)-14-hexanamido-13,21-dioxo-3,6,9,20-tetraoxa-16-thia-12,22-diazadotriacontyl)phosphonic    acid. (101)

In some embodiments, a compound of formula (J) is not one of thecompounds 16 to 22.

In some embodiments (e.g. those where the compound is adsorbed to analuminium salt), a compound of formula (J) is not one of compounds (17),(19) or (22).

The invention can use compounds of formula (J), or pharmaceuticallyacceptable salts or esters thereof.

Compounds Useful with the Invention

In general, and subject to provisos mentioned herein, TLR agonistsuseful with the invention are represented by formula (A1):

wherein:

R^(X) and R^(Y) are independently selected from H and C₁-C₆ alkyl;

-   -   X is selected from a covalent bond, O and NH;    -   Y is selected from a covalent bond, O, C(O), S and NH;    -   L is a linker e.g. selected from, C₁-C₆alkylene,        C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxy and        —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1        to 4 substituents independently selected from halo, OH,        C₁-C₄alkyl, —OP(O)(OH)₂ and —P(O)(OH)₂;    -   each p is independently selected from 1, 2, 3, 4, 5 and 6;    -   q is selected from 1, 2, 3 and 4;    -   n is selected from 1, 2 and 3; and    -   A is a TLR agonist moiety.

In one embodiment, the TLR agonist according to formula (A1) is asfollows: R^(X) and R^(Y) are H; X is O; L is selected from C₁-C₆alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substitutedwith 1 to 2 halogen atoms; p is selected from 1, 2 and 3; q is selectedfrom 1 and 2; and n is 1. Thus in these embodiments the adsorptivemoiety comprises a phosphate group.

In other embodiments, the TLR agonist according to formula (A1) is asfollows: R^(X) and R^(Y) are H; X is a covalent bond; L is selected fromC₁-C₆ alkylene and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 2 halogen atoms; p is selected from 1, 2 or 3; qis selected from 1 or 2; and n is 1. Thus in these embodiments theadsorptive moiety comprises a phosphonate group.

Useful ‘A’ moieties for formula (A1) include, but are not limited to,radicals of any of the following compounds, defined herein or asdisclosed in references 1-7 and 9-27:

Compounds of formula (I) or (II) as defined above;

In some embodiments, the TLR agonist moiety ‘A’ has a molecular weightof less than 1000 Da. In some embodiments, the TLR agonist of formula(A1) has a molecular weight of less than 1000 Da.

Preferred TLR agonists are water-soluble. Thus they can form ahomogenous solution when mixed in an aqueous buffer with water at pH 7at 25° C. and 1 atmosphere pressure to give a solution which has aconcentration of at least 50 μg/ml. The term “water-soluble” thusexcludes substances that are only sparingly soluble under theseconditions.

In addition to being water-soluble at pH 7, preferred water-soluble TLRagonists are soluble in water at a pH between 4 and 10 e.g. between 5and 9, between 6 and 8, or preferably between 6.5 and 7.5.

Adsorptive Moleties

The adsorptive moieties referred to above are functional groups whichare capable of adsorbing to an insoluble metal salt (for example to aninsoluble aluminium salt, such as aluminium oxyhydroxide) e.g. by ligandexchange or any other suitable mechanism. Thus the adsorptive moiety canendow an active compound (e.g. the TLR agonist) with the ability to beadsorbed to an insoluble metal salt.

Ligand exchange is a mechanism of adsorption whereby chemical moietieson an immunopotentiator compound exchange with chemical moieties on thesurface of an insoluble metal salt, thereby resulting in adsorption ofthe immunopotentiator to the surface of the metal salt. This is themajor mechanism of adsorption for phosphorylated proteins such as HBsAg[28,29]. Thus an immunopotentiator can comprise an adsorptive moietysuch as a phosphonate group, which can exchange with groups on thesurface of the salt, such as hydroxyl groups on aluminium oxyhydroxide.In some embodiments, however, adsorption can take place by any othersuitable mechanism e.g. by electrostatic or hydrophobic means [30](or bya combination of mechanisms). In some embodiments actual physicochemicaladsorption may not occur and a TLR agonist may instead be trapped invoid spaces within aggregates of the metal salt (e.g. as discussed inreference 31), but this entrapment can be enhanced by the presence ofthe adsorptive group. In other embodiments there may be a mixture ofadsorbed and entrapped TLR agonist.

Phosphorus-containing adsorptive moieties are particularly useful, andso an adsorptive moiety may comprise a phosphate, a phosphonate, aphosphinate, a phosphonite, a phosphinite, etc. One useful adsorptivemoiety comprises at least one phosphate group. A preferred adsorptivemoiety comprises at least one phosphonate group.

A group of useful phosphorus-containing adsorptive moieties are shown in[square brackets] in formula A1 and a TLR agonist may thus include aphosphate (optionally substituted) or phosphonate (optionallysubstituted) by which it can adsorb to an insoluble metal salt.

Phosphorous-containing groups employed with the invention may exist in anumber of protonated and deprotonated forms depending on the pH of thesurrounding environment, for example the pH of the solvent in which theyare dissolved. Therefore, although a particular form may be illustratedit is intended, unless otherwise mentioned, for these illustrations tomerely be representative and not limiting to a specific protonated ordeprotonated form.

For example, in the case of a phosphate group, this has been illustratedas —OP(O)(OH)₂ but the definition includes the protonated forms—[OP(O)(OH₂)(OH)]⁺ and —[OP(O)(OH₂)₂]²⁺ that may exist at a pH below 7,and the deprotonated forms —[OP(O)(OH)(O)]⁺ and [OP(O)(O)₂]²⁺ that mayexist at a pH above 7.

Compounds disclosed herein can exist as pharmaceutically acceptablesalts. Thus, the compounds may be used in the form of theirpharmaceutically acceptable salts i.e. physiologically ortoxicologically tolerable salt (which includes, when appropriate,pharmaceutically acceptable base addition salts and pharmaceuticallyacceptable acid addition salts).

Alternatives to phosphorus-containing functional groups to provide anadsorptive TLR agonist are nitrate and/or sulphate groups.

Compounds useful with the invention may include a single adsorptivemoiety, or may include more than one e.g. between 2 and 15 adsorptivemoieties. Typically a compound will include 1, 2 or 3 adsorptivemoieties.

Insoluble Metal Salts

As disclosed herein, immunopotentiators can adsorb to insoluble metalsalts, thereby forming an adsorbed complex. For instance, they can beadsorbed to insoluble calcium salts (e.g. calcium phosphate) or,preferably, to insoluble aluminium salts. Such aluminium salts have along history of use in vaccines. Aluminium salts which include hydroxideions are the preferred insoluble metal salts for use with the presentinvention.

Thus the invention provides various embodiments in which a TLR agonistis adsorbed to such insoluble salts e.g. a compound of formula (I),(II), (III), (I-A), (B), (C), (D), (E), (F), (G), (H) or (J).

Useful aluminium salts include, but are not limited to, aluminiumhydroxide, aluminium oxyhydroxide, and aluminium hydroxyphosphates(including aluminium hydroxyphosphate sulfate). Such salts are describede.g. in chapters 8 & 9 of reference 32.

Preferred salts for adsorption of immunopotentiators are aluminiumoxyhydroxides and/or aluminium hydroxyphosphate. These have surfacehydroxyl moieties which can readily undergo ligand exchange withphosphorus-containing groups (e.g. phosphates, phosphonates) to providestable adsorption.

The adjuvants commonly known as “aluminium hydroxide” are typicallyaluminium oxyhydroxide salts, which are usually at least partiallycrystalline. Aluminium oxyhydroxide, which can be represented by theformula AlO(OH), can be distinguished from other aluminium compounds,such as aluminium hydroxide Al(OH)₃, by infrared (IR) spectroscopy, inparticular by the presence of an adsorption band at 1070 cm⁻¹ and astrong shoulder at 3090-3100 cm⁻¹ (chapter 9 of ref. 32). The degree ofcrystallinity of an aluminium hydroxide adjuvant is reflected by thewidth of the diffraction band at half height (WHH), withpoorly-crystalline particles showing greater line broadening due tosmaller crystallite sizes. The surface area increases as WHH increases,and adjuvants with higher WHH values have been seen to have greatercapacity for antigen adsorption. A fibrous morphology (e.g. as seen intransmission electron micrographs) is typical for aluminium hydroxideadjuvants e.g. with needle-like particles with diameters about 2 nm. Thepi of aluminium hydroxide adjuvants is typically about 11 i.e. theadjuvant itself has a positive surface charge at physiological pH.Adsorptive capacities of between 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH7.4 have been reported for aluminium hydroxide adjuvants.

The adjuvants commonly known as “aluminium phosphate” are typicallyaluminium hydroxyphosphates, often also containing a small amount ofsulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtainedby precipitation, and the reaction conditions and concentrations duringprecipitation influence the degree of substitution of phosphate forhydroxyl in the salt. Hydroxyphosphates generally have a PO₄/Al molarratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished fromstrict AlPO₄ by the presence of hydroxyl groups. For example, an IRspectrum band at 3164 cm⁻¹ (e.g. when heated to 200° C.) indicates thepresence of structural hydroxyls (chapter 9 of reference 32).

The PO₄/Al³⁺ molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95±0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate willgenerally be particulate (e.g. plate-like morphology as seen intransmission electron micrographs, with primary particles in the rangeof 50 nm). Typical diameters of the particles are in the range 0.5-20 μm(e.g. about 5-10 μm) after any antigen adsorption. Adsorptive capacitiesof between 0.7-1.5 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reportedfor aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inverselyrelated to the degree of substitution of phosphate for hydroxyl, andthis degree of substitution can vary depending on reaction conditionsand concentration of reactants used for preparing the salt byprecipitation. PZC is also altered by changing the concentration of freephosphate ions in solution (more phosphate=more acidic PZC) or by addinga buffer such as a histidine buffer (makes PZC more basic). Aluminiumphosphates used according to the invention will generally have a PZC ofbetween 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

In solution both aluminium phosphate and hydroxide adjuvants tend toform stable porous aggregates 1-10 μm in diameter [33].

A composition including an TLR agonist of the invention adsorbed to ametal salt can also include a buffer (e.g. a phosphate or a histidine ora Tris buffer). When such a composition includes a phosphate buffer,however, it is preferred that the concentration of phosphate ions in thebuffer should be less than 50 mM e.g. <40 mM, <30 mM, <20 mM, <10 mM, or<5 mM, or between 1-15 mM. In embodiments of the fifteenth aspect,however, using a phosphate buffer is not permitted.

Because of the insolubility of adsorptive metal salts which are usefulwith the invention, compositions containing adsorbed immunopotentiatorswill generally be suspensions having a cloudy appearance. This can maskcontaminating bacterial growth and so a composition of the invention mayinclude a preservative such as thiomersal or 2-phenoxyethanol. It ispreferred that a composition should be substantially free from (e.g. <10μg/ml) mercurial material e.g. thiomersal-free. Vaccines containing nomercury are more preferred.

A composition can include a mixture of both an aluminium oxyhydroxideand an aluminium hydroxyphosphate, and an immunopotentiator may beadsorbed to one or both of these salts.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 10 mg/ml e.g. ≦5 mg/ml, ≦4 mg/ml, ≦3mg/ml, ≦2 mg/ml, ≦1 mg/ml, etc. A preferred range of Al⁺⁺⁺ in acomposition of the invention is between 0.3 and 1 mg/ml or between0.3-0.5 mg/mi. A maximum of 0.85 mg/dose is preferred. Because theinclusion of a TLR agonist can improve the adjuvant effect of aluminiumsalts then the invention advantageously permits lower amounts of Al⁺⁺⁺per dose, and so a composition of the invention can usefully includebetween 10 and 250 μg of Al⁺⁺⁺ per unit dose. Current pediatric vaccinestypically include at least 300 μg Al⁺⁺⁺. In concentration terms, acomposition of the invention may have an Al⁺⁺⁺ concentration between 10and 500 μg/ml e.g. between 10-300 μg/ml, between 10-2001 μg/ml, orbetween 10-100 μg/ml.

In general, when a composition includes both a TLR agonist and analuminium salt, the weight ratio of agonist to Al⁺⁺⁺ will be less than5:1 e.g. less than 4:1, less than 3:1, less than 2:1, or less than 1:1.Thus, for example, with an Al⁺⁺⁺ concentration of 0.5 mg/ml the maximumconcentration of TLR agonist would be 2.5 mg/ml. But higher or lowerlevels can be used; a lower mass of TLR agonist than of Al⁺⁺⁺ is typicale.g. per dose, 100 g of TLR agonist with 0.2 mg Al⁺⁺⁺.

Where a composition includes a TLR agonist and an insoluble metal salt,it is preferred that at least 50% (by mass) of the immunopotentiator inthe composition is adsorbed to the metal salt e.g. ≧60%, ≧70%, 280%,≧85%, 290%, ≧92%, 194%, ≧95%, 296%, ≧97%, ≧98%, ≧99%, or even 100%. Aminimum of 80% adsorption is typical, and at least 90% or 95% ispreferred.

As discussed above, as a result of adsorption to an insoluble metal saltcan modify the in vivo behaviour of SMIPs. Thus an adsorbed SMIP candisplay a longer residence time (e.g. at least 2× longer) in muscleafter intramuscular injection, relative to the same SMIP injected innon-adsorbed form. Some clearance can occur, but a detectable portion ofthe injected SMIP will still be present. Thus, for instance, an adsorbedSMIP can, when injected intramuscularly, still be present in theinjected muscle at least 12 hours later e.g. 24 hours later.

In some embodiments, an adsorbed SMIP can display a lower peak serumconcentration, relative to the same SMIP injected in non-adsorbed form.This peak is usually expressed as a Cmax value. For instance, anadsorbed SMIP can, when injected intramuscularly, have a lower serumCmax value than the same SMIP when injected intramuscularly innon-adsorbed form (e.g. <95% of the non-adsorbed Cmax, <80% of thenon-adsorbed Cmax, <50% of the non-adsorbed Cmax, or even <30% of thenon-adsorbed Cmax).

In some embodiments, an adsorbed SMIP can display a lower total systemicexposure after injection, relative to the same SMIP injected innon-adsorbed form. Levels of systemic exposure are usually expressed asAUC (area under the concentration-time curve) values (e.g. in nM-hr).Advantageously, for instance, an adsorbed SMIP can, when injectedintramuscularly, have a lower serum AUC value in the 24 hours followinginjection than the same SMIP when injected intramuscularly innon-adsorbed form (e.g. <90% of the non-adsorbed AUC, <80% of thenon-adsorbed AUC, or even <50% of the non-adsorbed AUC, etc.).

Immunogens

Adsorbed immunopotentiators of the invention are useful duringimmunisation. An adsorbed complex of the invention can thus be used inconjunction with one or more immunogen(s). The complex and immunogen(s)can be provided as an admixture, or can be provided separately for useafter mixing. In some embodiments, an immunopotentiators of theinvention can be combined with an immunogen in the absence of aninsoluble metal salt, and can thereafter either be administered to amammal or can be combined with an insoluble metal salt for lateradministration to a mammal.

The invention can be used with a wide range of immunogens, for treatingor protecting against a wide range of diseases. The immunogen may elicitan immune response that protects against a viral disease (e.g. due to anenveloped or non-enveloped virus), a bacterial disease (e.g. due to aGram negative or a Gram positive bacterium), a fungal disease, aparasitic disease, an auto-immune disease, or any other disease. Theimmunogen may also be useful in immunotherapy e.g. for treating atumour/cancer, Alzheimer's disease, or an addiction.

The immunogen may take various forms e.g. a whole organism, anouter-membrane vesicle, a polypeptidc, a saccharide, a liposaccharide, aconjugate (e.g. of a carrier and a hapten, or of a carrier and asaccharide or liposaccharide), etc. Where the immunogen is apolypeptide, it will typically be a surface polypeptide e.g. an adhesin,a hemagglutinin, an envelope glycoprotein, a spike glycoprotein, etc.

The immunogen may elicit an immune response against an influenza virus,including influenza A and B viruses. Various forms of influenza virusimmunogen are currently available, typically based either on live virusor on inactivated virus. Inactivated vaccines may be based on wholevirions, split virions, or on purified surface antigens. Influenzaantigens can also be presented in the form of virosomes. Hemagglutininis the main immunogen in current inactivated vaccines, and vaccine dosesare standardised by reference to HA levels, typically measured by SRID.Existing vaccines typically contain about 15 μg of HA per strain,although lower doses can be used e.g. for children, or in pandemicsituations, or when using an adjuvant. Fractional doses such as ½ (i.e.7.5 μg HA per strain), ¼ and ⅛ have been used, as have higher doses(e.g. 3× or 9× doses [34,35]). Thus compositions may include between 0.1and 150 μg of HA per influenza strain, preferably between 0.1 and 50 μge.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.51 μg, 0.5-51 μg, etc.Particular doses include e.g. about 45, about 30, about 15, about 10,about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc.per strain. It is usual to include substantially the same mass of HA foreach strain included in the vaccine e.g. such that the HA mass for eachstrain is within 10% of the mean HA mass per strain, and preferablywithin 5% of the mean. For live vaccines, dosing is measured by mediantissue culture infectious dose (TCID₅₀) rather than HA content, and aTCID₅₀ of between 10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) perstrain is typical. Rather than use SPF eggs as the substrate for viralgrowth, where virus is harvested from infected allantoic fluids of hens'eggs, cell lines that support influenza virus replication may be used.The cell line will typically be of mammalian origin e.g. MDCK.

Influenza A virus immunogens may be from any suitable HA subtype straine.g. H1, H3, H5, H7, H9 etc., such as a H1N1, H3N2 and/or H5N1 strain.

The immunogen may elicit an immune response against a Candida fungussuch as C. alblcans. For instance, the immunogen may be a β-glucan,which may be conjugated to a carrier protein. The glucan may includeβ-1,3 and/or β-1,6 linkages. Suitable immunogens include those disclosedin references 36 & 37.

The immunogen may elicit an immune response against a Streptococcusbacterium, including S. agalactiae, S. pneumoniae and S. pyogenes. Forinstance, the immunogen may be a capsular saccharide, which may beconjugated to a carrier protein. For S. agalactiae the saccharide may befrom one or more of serotypes Ia, Ib, II, III, and/or V. For S.pneumoniae the saccharide may be from one or more of serotypes 1, 3, 4,5, 6B, 7F, 9V, 14, 18C, 19F, and/or 23F. In addition to (or in place of)capsular saccharide immunogen(s), polypeptide immunogens may be used toelicit a protective anti-streptococcal immune response e.g. comprisingRrgB, as disclosed in reference 38.

The immunogen may elicit an immune response against a Staphylococcusbacterium, including S. aureus or S. epidermidis. For instance, theimmunogen may comprise an IsdA antigen, an IsdB antigen, a ClfA antigen,a ClfB antigen, a SdrD antigen, a Spa antigen, an EsxA antigen, an EsxBantigen, a Sta006 antigen, a hemolysin, and/or a Sta011 antigen.Suitable S. aureus immunogens and their combinations are disclosed inreference 39.

The immunogen may elicit an immune response against a meningococcalbacterium (Neisseria meningitidis). For instance, the immunogen may be acapsular saccharide, which may be conjugated to a carrier protein.Capsular saccharides are particularly useful for protecting againstmeningococcal serogroups A, C, W135 and/or Y. In addition to (or inplace of) capsular saccharide immunogen(s), polypeptide immunogensand/or outer membrane vesicles may be used to elicit a protectiveanti-meningococcal immune response, particularly for use againstserogroup B e.g. as disclosed in reference 40. A typical amount ofcapsular saccharide per unit dose of a vaccine is between 2.5-10 μg,although lower doses can be used with the invention due to theantigen-sparing nature of the adjuvants.

The immunogen may elicit an immune response against a hepatitis virus,such as a hepatitis A virus, a hepatitis B virus, a hepatitis C virusand/or a hepatitis E virus. For instance, the immunogen may be hepatitisB virus surface antigen (HBsAg). A typical amount of HBsAg per unit doseof a vaccine is between 5-20 μg, but lower doses can be used with theinvention due to the antigen-sparing nature of the adjuvants.

The immunogen may elicit an immune response against a respiratorysyncytial virus. Immunogens may be from a group A RSV and/or a group BRSV. Suitable immunogens may comprise the F and/or G glycoproteins orfragments thereof e.g. as disclosed in references 41 & 42.

The immunogen may elicit an immune response against a Chlamydiabacterium, including C. trachomatis and C. pneumoniae. Suitableimmunogens include those disclosed in references 43-49.

The immunogen may elicit an immune response against an Escherichia colibacterium, including extraintestinal pathogenic strains. Suitableimmunogens include those disclosed in references 50-52.

The immunogen may elicit an immune response against a coronavirus, suchas the human SARS coronavirus. Suitable immunogens may comprise thespike glycoprotein.

The immunogen may elicit an immune response against a Helicobacterpylori bacterium. Suitable immunogens include CagA [53-56], VacA[57,58], and/or NAP [59-61].

The immunogen may elicit an immune response against a Corynebacteriumdiphtheriae bacterium.

Suitable immunogens include diphtheria toxoid (“DT”). A typical amountof DT per unit dose of a pediatric vaccine is between 15-30 Lf (“limesflocculating dose”), although lower doses can be used with the inventiondue to the antigen-sparing nature of the adjuvants. Lower amounts arealso typical in adolescent or adult booster vaccines e.g. between 1-10Lf/dose.

The immunogen may elicit an immune response against a Clostridium tetanibacterium. Suitable immunogens include tetanus toxoid (“TT”). A typicalamount of TT per unit dose of a pediatric vaccine is between 5-15 Lf(“limes flocculating dose”), although lower doses can be used with theinvention due to the antigen-sparing nature of the adjuvants. Loweramounts are also typical in adolescent or adult booster vaccines e.g.between 1-5 Lf/dose.

The immunogen may elicit an immune response against a Bordetellaperlussis bacterium. Pertussis antigens are either cellular (whole cell,in the form of inactivated B. pertussis cells; ‘wP’) or acellular(‘aP’). Where acellular antigens are used, one, two or (preferably)three of the following antigens are included: (1) detoxified pertussistoxin (pertussis toxoid, or ‘PT’); (2) filamentous hemagglutinin(‘FHA’); (3) pertactin (also known as the ‘69 kiloDalton outer membraneprotein’). The PT may be chemically detoxified or may be a mutant PT inwhich enzymatic activity has been reduced by mutagenesis [62]e.g. the9K/129G double mutant [63]. As well as PT, FHA and pertactin, it is alsopossible to include fimbriae (e.g. agglutinogens 2 and 3) in anacellular pertussis antigen component. A typical amount of PT in apediatric vaccine is 10-30 μg/dose. A typical amount of FHA in apediatric vaccine is 15-30 μg/dose. A typical amount of pertactin in apediatric vaccine is 2-10 μg/dose. Lower doses can be used with theinvention due to the antigen-sparing nature of the adjuvants. Loweramounts are also typical in booster vaccines e.g. ˜3 times lower.

The immunogen may elicit an immune response against a Haemophilusinfluenzae type B bacterium (“Hib”). Suitable immunogens includeconjugates of the Hib capsular saccharide (“PRP) e.g. conjugated totetanus toxoid, diphtheria toxoid, the CRM 197 derivative of diphtheriatoxoid, H. influenzae protein D, and an outer membrane protein complexfrom serogroup B meningococcus. A typical amount of Hib conjugate(measured as saccharide) is between 2.5-15 μg per dose, although lowerdoses can be used with the invention due to the antigen-sparing natureof the adjuvants.

The immunogen may elicit an immune response against a poliovirus.Suitable immunogens include inactivated viruses. A typical compositionwill include three poliovirus antigens—poliovirus Type 1 (e.g. Mahoneystrain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirus Type 3(e.g. Saukett strain). A typical amount of poliovirus per dose is 40 DU(“D-antigen unit”) for Type 1, 8 DU for Type 2, and 32 DU for Type 3,although lower doses can be used with the invention due to theantigen-sparing nature of the adjuvants.

The immunogen may elicit an immune response against a cytomegalovirus(‘CMV’). For example, the immunogen may be a recombinant glycoprotein Be.g. the soluble antigen used in reference 64.

The immunogen may elicit an immune response against a humanimmunodeficiency virus e.g. against HIV-1 or HIV-2. For example, theimmunogen may be a HIV envelope glycoprotein. For instance, engineeredenvelope glycoproteins are available, such as gp140, which can formoligomers (referred to as ‘o-gp140’). The gp140 polypeptide includes thegp120 sequence and the ectodomain of gp41 [65], and has been reported tobe a better immunogen than gp120 [66]. Thus a useful envelopeglycoprotein may include a portion of gp41 but not include itstransmembrane domain. The gp140 form of the envelope glycoprotein canhave its V2 loop deleted, to give gp140ΔV2 mutants, and such delectionshave been reported to improve immunogenicity. The ΔV2 mutants of gp140have been shown to form trimers [67].

The immunogen may elicit an immune response against rabies virus. Asuitable immunogen is an inactivated rabies virus [68, RabAvert™].

The immunogen may elicit an immune response against a humanpapillomavirus. Useful immunogens are L1 capsid proteins, which canassemble to form structures known as virus-like particles (VLPs). TheVLPs can be produced by recombinant expression of L1 in yeast cells(e.g. in S. cerevisiae) or in insect cells (e.g. in Spodoptera cells,such as S. frugiperda, or in Drosophila cells).

For yeast cells, plasmid vectors can carry the L1 gene(s); for insectcells, baculovirus vectors can carry the L1 gene(s). More preferably,the composition includes L1 VLPs from both HPV-16 and HPV-18 strains.This bivalent combination has been shown to be highly effective [69]. Inaddition to HPV-16 and HPV-18 strains, it is also possible to include L1VLPs from HPV-6 and HPV-11 strains.

The immunogen may elicit an immune response against a tumour antigen,such as MAGE-1, MAGE-2, MAGE-3 (MAGE-A3), MART-1/Melan A, tyrosinase,gp100, TRP-2, etc. The immunogen may elicit an immunotherapeuticresponse against lung cancer, melanoma, breast cancer, prostate cancer,etc.

The immunogen may elicit an immune response against a hapten conjugatedto a carrier protein, where the hapten is a drug of abuse [70]. Examplesinclude, but are not limited to, opiates, marijuana, amphetamines,cocaine, barbituates, glutethimide, methyprylon, chloral hydrate,methaqualone, benzodiazepines, LSD, nicotine, anticholinergic drugs,antipsychotic drugs, tryptamine, other psychomimetic drugs, sedatives,phencyclidine, psilocybine, volatile nitrite, and other drugs inducingphysical and/or psychological dependence.

Various other immunogens may be used.

Compositions for immunisation against Neisseria meningitides Theinvention is particularly useful for immunising against meningococcuse.g. against serogroup B.

One preferred immunogenic composition of the invention comprises: (i) analuminium hydroxide adjuvant; (ii) compound 1, 2 or 5 herein; (iii) afirst polypeptide comprising SEQ ID NO: 1; (iv) a second polypeptidecomprising SEQ ID NO: 2; and (v) a third polypeptide comprising SEQ IDNO: 3; wherein the compound of (ii) is adsorbed to the aluminiumhydroxide.

Another preferred immunogenic composition of the invention comprises:(i) an aluminium hydroxide adjuvant; (ii) compound 1, 2 or 5 herein;(iii) a first polypeptide comprising SEQ ID NO: 1; (iv) a secondpolypeptide comprising SEQ ID NO: 2; and (v) a third polypeptidecomprising SEQ ID NO: 4; wherein the compound of (ii) is adsorbed to thealuminium hydroxide.

Another preferred immunogenic composition of the invention comprises:(i) an aluminium hydroxide adjuvant; (ii) compound 1, 2 or 5 herein;(iii) a first polypeptide comprising SEQ ID NO: 1; (iv) a secondpolypeptide comprising SEQ ID NO: 2; and (v) a third polypeptidecomprising SEQ ID NO: 5; wherein the compound of (ii) is adsorbed to thealuminium hydroxide.

Any of the first, second and/or third polypeptides can differ from therelevant SEQ ID NO: 1, 2, 3, 4 or 5 by up to 3 amino acids, providedthat the polypeptide can still elicit antibodies which bind to apolypeptide which consists of SEQ ID NO: 1, 2, 3, 4 or 5, asappropriate.

Ideally, 1 2 or 3 of the first second and/or third polypeptides is/arealso adsorbed to the aluminium hydroxide. These polypeptides aredisclosed in more detail in references 40, 71 and 91. The compositionmay include 5-100 μg of each polypeptide. The composition ideally doesnot include any bacterial outer membrane vesicles.

The composition may include from 5-100 μg of compound 1, 2 or 5. Forexample, it may include from 5-100 μg of compound 2, or it may includefrom 5-100 μg of compound 5.

The composition may include a histidine buffer e.g. a 10 mM histidinebuffer. It may include sucrose and/or sodium chloride. It may beadministered in a dosage volume of 0.5 ml e.g. for intramuscularinjection.

Further immunogenic compositions of the invention may comprise: (i) analuminium hydroxide adjuvant; (ii) a TLR7 agonist of formula (I-A) or offormula (IV); (iii) a meningococcal factor H binding protein antigen,provided that this antigen is not a fusion protein having an amino acidsequence comprising SEQ ID NO: 8 from reference 73. The factor H bindingprotein antigen can be adsorbed to the aluminium hydroxide too.

Compositions with Multiple Different Immunogens

According to a twenty-fifth aspect, the invention provides a compositioncomprising an adjuvant complex of the invention in combination with atleast two different immunogens.

The invention also provides a kit comprising (i) an adjuvant complex ina first container and (ii) at least one immunogen in a second container.The first container can optionally include at least one immunogen inaddition to the complex.

The TLR agonist in the adjuvant complex can be any agonist as disclosedherein.

In some embodiments the TLR agonist is a TLR7 agonist according to anyof formulae (B), (C), (D), (E), or (H). In some embodiments the TLRagonist is a TLR2 agonist according to formula (F) or (J). In someembodiments the TLR agonist is a TLR8 agonist according to formula (G).

In some embodiments the TLR agonist is not a TLR4 agonist. In someembodiments the TLR agonist is not a TLR9 agonist. In some embodimentsthe TLR agonist is not a compound according to formula (I) as definedherein. In some embodiments the TLR agonist is not a compound accordingto formula (II) as defined herein. In some embodiments the TLR agonistis not a compound according to formula (III) as defined herein. In someembodiments the TLR agonist is not a compound according to Formula (I-A)as defined herein.

In preferred embodiments the TLR agonist is a TLR7 agonist according toformula (B). Specific TLR7 agonists of interest include compounds IA to27A in Table A on pages 79-84 of reference 72.

Where the TLR agonist is a TLR7 agonist according to formula (B), the“at least two different immunogens” in some embodiments does not consistof: (i) a combination of a measles virus immunogen, a mumps virusimmunogen, and a rubella virus immunogen; (ii) a combination of ameasles virus immunogen, a mumps virus immunogen, a rubella virusimmunogen, and a varicella virus immunogen; (iii) a diphtheria vaccine,a tetanus vaccine, and a pertussis vaccine; (iv) a tetravalentcombination of conjugates from meningococcus serogroups A, C, W135 andY; (v) a combination of bacterial antigens from serogroups A, B, C, W135and/or Y of N. meningitidis; (vi) a combination including antigens fromtwo or more different strains of influenza viruses; (vii) a combinationof outer-membrane vesicles from serogroups A, C, W135, Y, X and/or B ofN. meningitidis; (viii) a combination of saccharides from differentpneumococcal serotypes; (ix) a combination of Moraxella catarrhalisantigens; (x) a combination of Bordetella pertussis holotoxin,filamentous haemagglutinin, pertactin and/or agglutinogens 2 and 3; (xi)a combination of multiple different polypeptide antigens from N.meningitidis.

Where the TLR agonist is a TLR7 agonist which is Compound 1, 2, 3, 4, 5or 13 herein then the “at least two different immunogens” in someembodiments does not consist of a combination of multiple differentpolypeptide antigens from N. meningitidis such as the combinationdisclosed in references 40 and 73.

The “at least two different immunogens” can include at least onebacterial antigen and at least one viral antigen.

If the “at least two different immunogens” include only bacterialimmunogens then they ideally include immunogens for at least twodifferent species of bacteria (thus, for instance, excluding acombination of different meningococcal capsular saccharides, as theseare all from a single species).

The “at least two different immunogens” should not be conjugated to eachother. Thus a conjugate of a Hib saccharide and a tetanus toxoid is not“at least two different immunogens” as used herein.

Preferred embodiments of “at least two different immunogens” includecompositions including: (i) a diphtheria toxoid, a tetanus toxoid, andan acellular pertussis antigen e.g. comprising a pertussis toxoid,filamentous hemagglutinin and/or pertactin; (ii) a diphtheria toxoid, atetanus toxoid, a pertussis antigen, and a H. influenzae type B capsularsaccharide conjugate; (iii) a diphtheria toxoid, a tetanus toxoid, apertussis antigen, and a hepatitis B virus surface antigen; (iv) adiphtheria toxoid, a tetanus toxoid, a pertussis antigen, a hepatitis Bvirus surface antigen and a H. influenzae type B capsular saccharideconjugate; (v) a diphtheria toxoid, a tetanus toxoid, a pertussisantigen, and an inactivated poliovirus antigen; (vi) a diphtheriatoxoid, a tetanus toxoid, a pertussis antigen, a H. influenzae type Bcapsular saccharide conjugate, a hepatitis B virus surface antigen, andan inactivated poliovirus antigen; or (vii) a hepatitis A virus antigenand a hepatitis B virus antigen.

Where a composition includes an inactivated poliovirus antigen itpreferably includes antigens from each of poliovirus Type 1 (e.g.Mahoney strain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirusType 3 (e.g. Saukett strain).

Where a composition includes a pertussis antigen it ideally does notinclude whole inactivated B. pertussis cells i.e. it is ideally anacellular vaccine.

As well as including D, T, Pa, HBsAg, Hib and/or poliovirus antigens, acomposition of the invention may include further antigens e.g. fromfurther pathogens. For example, these antigens may be from N.meningitidis (one or more of serogroups A, B, C, W135 and/or Y) or S.pneumoniae. Thus a composition may include two or three of: (i) one ormore of D, T, Pa, HBsAg, Hib and/or poliovirus antigens; (ii) aconjugated capsular saccharide from one or more of meningococcalserogroups A, C, W135 and/or Y; (iii) a polypeptide antigen frommeningococcus, such as a fHbp.

Compositions of the invention which include multiple immunogenspreferably do not include any bacterial outer membrane vesicles.

TLR Agonism

The invention utilises TLR agonists which comprise an adsorptive moietyand a TLR agonist moiety. The adsorptive moiety confers the ability toadsorb to an insoluble metal salt (see above), whereas the TLR agonistmoiety confers the ability to agonise a Toll-like receptor. Typically aTLR agonist of the invention would thus function as a TLR agonist evenwithout its adsorptive moiety. Except where otherwise stated, TLRagonists of the invention can activate any of TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9 or TLR11.

Where the invention provides or refers to a compound as a TLR agonist,the compound is preferably an agonist of TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10 or TLR11.

From this group, a preferred sub-group is TLR1, TLR2, TLR3, TLR5, TLR6,TLR7, TLR8 and TLR11. A more preferred subgroup is TLR2, TLR7 and TLR8.Another preferred subgroup is TLR2 and TLR7.

Most preferably, a TLR agonist is an agonist of a human TLR.

A compound of the invention may be an agonist of human TLR1. A compoundof the invention may be an agonist of human TLR2. A compound of theinvention may be an agonist of human TLR3. A compound of the inventionmay be an agonist of human TLR4. A compound of the invention may be anagonist of human TLR5. A compound of the invention may be an agonist ofhuman TLR6. A compound of the invention may be an agonist of human TLR7.A compound of the invention may be an agonist of human TLR5. A compoundof the invention may be an agonist of human TLR9. A compound of theinvention may be an agonist of human TLR1.

Agonist activity of a compound against any particular Toll-like receptorcan be determined by standard assays. Companies such as Imgenex,Invivogen supply cell lines which are stably co-transfected with humanTLR genes and NFκB, plus suitable reporter genes, for measuring TLRactivation pathways. They are designed for sensitivity, broad workingrange dynamics and can be used for high-throughput screening.Constitutive expression of one or two specific TLRs is typical in suchcell lines. See also reference 74.

According to a twenty-sixth aspect, a composition includes first andsecond TLR agonists. These two agonists are different from each otherand they target different TLRs. Both agonists are adsorbed to analuminium salt. They may be co-adsorbed to an aluminium salt or they maybe separately adsorbed to aluminium salts (preferably the same salt e.g.both to aluminium hydroxide) and then combined. TLR combinations areknown from e.g. reference 75.

In Situ Precipitation Processes

According to a twenty-seventh aspect, the invention provides a processfor preparing an adjuvant complex, comprising steps of (i) preparing anaqueous mixture of a TLR agonist and a soluble aluminium salt; then (ii)adding a non-aluminium salt to the aqueous mixture in order to form aprecipitated aluminium salt to which the TLR agonist is adsorbed.

According to a twenty-eighth aspect, the invention provides a processfor preparing an immunogenic composition, comprising a step of mixing(i) an aqueous mixture of a TLR agonist and a soluble aluminium saltwith (ii) a buffered aqueous mixture of an immunogen, wherein the mixingstep causes precipitation of an aluminium salt to which the TLR agonistand the immunogen are adsorbed.

The invention also provides a process for preparing an immunogeniccomposition, comprising a step of mixing (i) an aqueous solution of asoluble aluminium salt with (ii) a buffered aqueous mixture of animmunogen and a TLR agonist, wherein the mixing step causesprecipitation of an aluminium salt to which the TLR agonist and theimmunogen are adsorbed.

The invention also provides a process for preparing an immunogeniccomposition, comprising a step of mixing (i) an aqueous solution of asoluble aluminium salt and an immunogen with (ii) a buffered aqueousmixture of a TLR agonist, wherein the mixing step causes precipitationof an aluminium salt to which the TLR agonist and the immunogen areadsorbed.

The invention also provides immunogenic compositions obtained orobtainable by these processes.

In these processes the soluble aluminium salt will typically be alum(KAl(SO₄)₂, typically as KAl(SO₄)₂.12H₂O) or aluminium chloride. Addingan alternative anion to this soluble salt can cause an aluminium saltadjuvant to precipitate in situ.

The alternative anion is typically added as part of a buffer. Thus, forinstance, if a phosphate buffer is added to the soluble aluminium saltthen an aluminium phosphate adjuvant can precipitate. The buffer willtypically be an acetate, carbonate, or phosphate buffer. Addition of thebuffer to an alum solution leads to precipitation of an amorphousaluminium hydroxy(buffer anion)sulfate e.g. aluminiumhydroxyphosphatesulfate (see chapter 9 of reference 32). The immunogenand the TLR agonist can adsorb to the precipitated salt.

The process may involve addition of a solution of a desired immunogen ina phosphate buffer. These processes can lead to compositions in whichimmunogcn and/or TLR agonist are adsorbed to an aluminiumhydroxyphosphatesulfate.

Analytical Assays

According to a twenty-ninth aspect, the invention provides an assay foranalysing an adjuvant complex which comprises a TLR agonist adsorbed toan insoluble metal salt, comprising steps of: (i) treating the complexto desorb TLR agonist from the insoluble metal salt; then (ii) detectingthe desorbed TLR agonist.

Various techniques are available for desorption, as required by step(i). For example, the complex can be treated with an adsorptive compoundwhich has higher affinity for the metal salt and which thus displacesthe adsorbed TLR agonist. Another method involves incubating the complexwith a high concentration of a phosphate buffer, such that the bufferions displace the adsorbed TLR agonist. Another method uses mixtures ofsalts and detergents e.g. see reference 76. Another method dissolves theinsoluble metal salt, thus destroying adsorption at the same time;suitable ways of dissolving such salts include e.g. citrate at anappropriate pH can be used to dissolve aluminium salt adjuvants, as canother polybasic or α-hydroxy-carboxylic acids.

Detection in step (ii) can be qualitative, quantitative, orsemi-quantitative. Thus the detection step can merely confirm thatdesorption has occurred, or can be used to quantify the amount ofagonist which had been adsorbed to the metal salt. Techniques such asHPLC can be used for quantitative detection of desorbed agonists.

In another aspect the invention provides an assay for analysing anadjuvant complex which comprises a TLR agonist adsorbed to an insolublemetal salt, comprising steps of: (i) treating the complex to desorb TLRagonist from the insoluble metal salt; then (ii) testing the biologicalactivity of the desorbed TLR agonist. Techniques for testing for theagonists' activity are well known in the art (see above). This assay,particularly when activity is analysed quantitatively, can be used toconfirm the stability of adsorbed material e.g. to check that the totalactivity of material which was adsorbed to the salts is retained afterdesorption. If all material is desorbed, but activity has been lost,this can indicate that the material has degraded while it was adsorbed.

Pharmaceutical Compositions and Products

The invention provides a pharmaceutical composition comprising a TLRagonist of the invention.

This composition can also include an insoluble metal salt and/or animmunogen.

The invention also provides a pharmaceutical composition comprising aTLR agonist of the invention and an insoluble metal salt. Thiscomposition can also include an immunogen.

The invention also provides an immunogenic pharmaceutical compositioncomprising a TLR agonist of the invention and an immunogen. Thiscomposition can also include an insoluble metal salt.

The invention also provides a method for preparing a pharmaceuticalcomposition, comprising a step of combining a TLR agonist of theinvention with one or more pharmaceutically acceptable excipients.

The invention also provides a method for preparing a pharmaceuticalcomposition, comprising a step of combining a complex of the inventionwith one or more pharmaceutically acceptable excipients.

Pharmaceutical compositions usually include components in addition tothe TLR agonist, insoluble metal salt and/or immunogen e.g. theytypically include one or more pharmaceutical carrier(s) and/orexcipient(s). A thorough discussion of such components is available inreference 77.

Pharmaceutical compositions are preferably in aqueous form, particularlyat the point of administration, but they can also be presented innon-aqueous liquid forms or in dried forms e.g. as gelatin capsules, oras lyophilisates, etc.

Pharmaceutical compositions may include one or more preservatives, suchas thiomersal or 2-phenoxyethanol. Mercury-free compositions arepreferred, and preservative-free vaccines can be prepared.

Pharmaceutical compositions can include a physiological salt, such as asodium salt e.g. to control tonicity. Sodium chloride (NaCl) is typical,which may be present at between 1 and 20 mg/ml e.g. 10±2 mg/ml or 9mg/ml. Other salts that may be present include potassium chloride,potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesiumchloride, calcium chloride, etc.

Pharmaceutical compositions can have an osmolality of between 200mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between290-310 mOsm/kg.

Pharmaceutical compositions may include compounds (with or without aninsoluble metal salt) in plain water (e.g. w.f.i.) but will usuallyinclude one or more buffers. Typical buffers include: a phosphate buffer(except in the fifteenth aspect); a Tris buffer; a borate buffer; asuccinate buffer; a histidine buffer (particularly with an aluminiumhydroxide adjuvant); or a citrate buffer. Buffer salt s will typicallybe included in the 5-20 mM range. If a phosphate buffer is used then theconcentration of phosphate ions should, in some embodiments, be <50 mM(see above) e.g. <10 mM.

Pharmaceutical compositions typically have a pH between 5.0 and 9.5 e.g.between 6.0 and 8.0.

Pharmaceutical compositions are preferably sterile.

Pharmaceutical compositions preferably non-pyrogenic e.g. containing <1EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EUper dose.

Pharmaceutical compositions are preferably gluten free.

Pharmaceutical compositions are suitable for administration to animal(and, in particular, human) patients, and thus include both human andveterinary uses. They may be used in a method of raising an immuneresponse in a patient, comprising the step of administering thecomposition to the patient. Compositions may be administered before asubject is exposed to a pathogen and/or after a subject is exposed to apathogen.

Pharmaceutical compositions may be prepared in unit dose form. In someembodiments a unit dose may have a volume of between 0.1-1.0 ml e.g.about 0.5 ml.

The invention also provides a delivery device (e.g. syringe, nebuliser,sprayer, inhaler, dermal patch, etc.) containing a pharmaceuticalcomposition of the invention e.g. containing a unit dose. This devicecan be used to administer the composition to a vertebrate subject.

The invention also provides a sterile container (e.g. a vial) containinga pharmaceutical composition of the invention e.g. containing a unitdose.

The invention also provides a unit dose of a pharmaceutical compositionof the invention.

The invention also provides a hermetically sealed container containing apharmaceutical composition of the invention. Suitable containers includee.g. a vial.

The invention also provides a kit comprising first and second kitcomponents, wherein: (i) the first kit component comprises an insolublemetal salt and an immunogen; and (ii) the second kit component comprisesa TLR agonist compound of the invention. The second component ideallydoes not include an insoluble metal salt and/or does not include animmunogen. The first and second components can be combined to provide acomposition suitable for administration to a subject.

The invention also provides a kit comprising first and second kitcomponents, wherein: (i) the first kit component comprises an insolublemetal salt and a TLR agonist compound of the invention; and (ii) thesecond kit component comprises an immunogen. The second componentideally does not include an insoluble metal salt and/or a TLR agonist.In some embodiments, the second component is lyophilised. The first andsecond components can be combined to provide a pharmaceuticalcomposition suitable for administration to a subject.

The invention also provides a kit comprising first and second kitcomponents, wherein: (i) the first kit component comprises an immunogenand a TLR agonist compound of the invention; and (ii) the second kitcomponent comprises an insoluble metal salt. The second componentideally does not include an immunogen and/or a TLR agonist. The firstand second components can be combined to provide a pharmaceuticalcomposition suitable for administration to a subject.

In some embodiments these kits comprise two vials. In other embodimentsthey comprise one ready-filled syringe and one vial, with the contentsof the syringe being mixed with the contents of the vial prior toinjection. A syringe/vial arrangement is useful where the vial'scontents are lyophilised. Usually, though, the first and second kitcomponents will both be in aqueous liquid form.

Pharmaceutical compositions of the invention may be prepared in variousforms. For example, the compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection canalso be prepared (e.g. a lyophilised composition or a spray-freeze driedcomposition). The composition may be prepared for topical administratione.g. as an ointment, cream or powder. The composition may be preparedfor oral administration e.g. as a tablet or capsule, as a spray, or as asyrup (optionally flavoured). The composition may be prepared forpulmonary administration e.g. by an inhaler, using a fine powder or aspray. The composition may be prepared as a suppository or pessary. Thecomposition may be prepared for nasal, aural or ocular administratione.g. as a spray or drops. The composition may be in kit form, designedsuch that a combined composition is reconstituted just prior toadministration to a patient. Such kits may comprise one or more antigensin liquid form and one or more lyophilised antigens. Injectables forintramuscular administration are typical.

Compositions comprise an effective amount of a TLR agonist i.e. anamount which, when administered to an individual, either in a singledose or as part of a series, is effective for enhancing the immuneresponse to a co-administered immunogen. This amount can vary dependingupon the health and physical condition of the individual to be treated,age, the taxonomic group of individual to be treated (e.g. non-humanprimate, primate, etc.), the capacity of the individual's immune systemto synthesise antibodies, the degree of protection desired, theformulation of the vaccine, the treating doctor's assessment of themedical situation, and other relevant factors. The amount will fall in arelatively broad range that can be determined through routine trials. Anamount of between 1-1000 μg/dose can be used e.g. from 5-100 μg per doseor from 10-100 μg per dose, and ideally ≦300 μg per dose e.g. about 5μg, 10 μg, 20 μg, 25 μg, 50 μg or 100 μg per dose. Thus theconcentration of a TLR agonist in a composition of the invention may befrom 2-2000 μg/ml e.g. from 10-200 μg/ml, or about 10, 20, 40, 50, 100or 200 μg/ml, and ideally ≦600 μg/ml. These ranges of doses, andspecific dosages, are particularly useful for compounds of formula (I-A)or (IV).

Methods of Treatment, and Administration f Immununogenic Compositions

The invention provides a method of raising an immune response in asubject, comprising the step of administering to the subject a TLRagonist, complex and/or composition of the invention.

The invention also provides a TLR agonist, complex and/or composition ofthe invention, for use in a method of raising an immune response in asubject.

The invention also provides the use of a TLR agonist or complex of theinvention in the manufacture of a medicament for raising an immuneresponse in a subject.

The invention also provides the use of (i) a TLR agonist as definedherein and (ii) an insoluble metal salt in the manufacture of amedicament for raising an immune response in a subject. Similarly, theinvention also provides the use of (i) a TLR agonist as defined herein(ii) an insoluble metal salt and (iii) an immunogen in the manufactureof a medicament (e.g. a vaccine) for raising an immune response in asubject.

The invention is suitable for raising immune responses in human ornon-human animal (in particular mammal) subjects. Compositions preparedaccording to the invention may be used to treat both children andadults.

The immune response stimulated by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses after immunisation are wellknown in the art.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. Administration of more than one dose(typically two doses) is particularly useful in immunologically naYvepatients. Multiple doses will typically be administered at least 1 weekapart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks,about 8 weeks, about 10 weeks, about 12 weeks, etc.).

Exemplary Compounds

Compounds 1 to 102 below contain adsorptive moieties coupled to corestructures that have been shown to be TLR agonists. Thus, thesecompounds can be adsorbed to insoluble metal salts as described herein.Thus the invention provides each of the following compounds:

The invention also provides a composition comprising two or more of saidcompounds 1 to 62. The invention also provides a composition comprisingtwo or more of said compounds 1 to 66. The invention also provides acomposition comprising two or more of said compounds 1 to 102.

Compounds 1 to 15, 23 to 51, 60 to 62, 67 to 71, and 102 arephospho-modified TLR7 agonists which are useful with the invention.

Compounds 16 to 22, 63 to 66 and 72 to 101 are phospho-modified TLR2agonists which are useful with the invention.

Compounds 52 to 59 are phospho-modified TLR8 agonists which are usefulwith the invention.

Other Biological Receptors

The invention is defined above by reference to TLR agonists, but it canbe more widely applied to other SMIPS which do not act via TLRs. Inparticular, SMIPs which may be used with the invention may agoniseC-type lectin receptors (CLRs) or CD1d rather than (or in addition to) aTLR. Thus the present disclosure includes the invention as describedabove with reference to TLR agonism, but wherein references to a TLRagonist (or similar) are replaced by reference either to a CLR agonistor to a CD1d agonist. Thus CLR or CD1d agonists can be modified tocontain at least one adsorptive moiety to provide the ability to adsorbto insoluble metal salt adjuvants.

CLR agonists include, but are not limited to, trehalose-6,6′-dimycolate(TDM), its synthetic analog D-(+)-trehalose-6,6′-dibehenate (TDB), andother 6,6′-diesters of trehalose and fatty acids. These trehalose esterscan readily be modified (e.g. as disclosed for the twenty-third aspect)to include an adsorptive group. The adsorptive group may be joined tothe immunopotentiator via a linker group, as discussed above. Themodified compound can adsorb to an insoluble metal salt. Thus theinvention can be applied to trehalose esters and diacyl trehaloses whichare CLR agonists. These agonists may have formula (T):

where R¹C(O)— and R²C(O)— are the same or different and are acyl groups,provided that (i) one of the monosaccharide rings (ii) R¹ or (iii) R²includes an adsorptive moiety. Suitable acyl groups may be saturated orunsaturated. They may be selected from the acyl residues of a mycolicacid, a corynomycolic acid, a 2-tetradecyl-3-hydroxyoctadecanoic acid, a2-eicosyl-3-hydroxytetracosanoic acid, a bourgeanic acid, a behenicacid, a palmitic acid, etc. Useful mycolic acids include alpha-,methoxy-, and keto-mycolic acids, in cis- and or trans-forms.

CD1d agonists include, but are not limited to, α-glycosylceramides[78-87] such as α-galactosylceramides. These can readily be modified(e.g. as disclosed for the twenty-third aspect) to include an adsorptivegroup. The adsorptive group may be joined to the immunopotentiator via alinker group, as discussed above. The modified compound can adsorb to aninsoluble metal salt. Thus the invention can be applied toglycosylceramides which are CD1d agonists, includingα-galactosylceramide, phytosphingosine-containing α-glycosylceramides,[(2S,3S,4R)-1-O-(α-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol],OCH, KRN7000 CRONY-101, 3″-O-sulfo-galactosylccramide, etc., providedthat the CD1d agonist includes an adsorptive group.

Chemical Groups

Unless specifically defined elsewhere, the chemical groups discussedherein have the following meaning when used in present specification:

The term “alkyl” includes saturated hydrocarbon residues including:

-   -   linear groups up to 10 atoms (C₁-C₁₀), or of up to 6 atoms        (C₁-C₆), or of up to 4 atoms (C₁-C₄). Examples of such alkyl        groups include, but are not limited, to C₁-methyl, C₂-ethyl,        C₃-propyl and C₄-n-butyl.    -   branched groups of between 3 and 10 atoms (C₃-C₁₀), or of up to        7 atoms (C₃-C₇), or of up to 4 atoms (C₃-C₄). Examples of such        alkyl groups include, but are not limited to, C₃-iso-propyl,        C₄-sec-butyl, C₄-iso-butyl, C₄-tert-butyl and C₅-neo-pentyl.

The term “alkylene” refers to the divalent hydrocarbon radical derivedfrom an alkyl group, and shall be construed in accordance with thedefinition above.

The term “alkenyl” includes monounsaturated hydrocarbon residuesincluding:

-   -   linear groups of between 2 and 6 atoms (C₂-C₆). Examples of such        alkenyl groups include, but are not limited to, C₂-vinyl,        C₃-1-propenyl, C₃-allyl, C₄-2-butenyl    -   branched groups of between 3 and 8 atoms (C₃-C₅). Examples of        such alkenyl groups include, but are not limited to,        C₄-2-methyl-2-propenyl and C₆-2,3-dimethyl-2-butenyl.

The term alkenylene refers to the divalent hydrocarbon radical derivedfrom an alkenyl group, and shall be construed in accordance with thedefinition above.

The term “alkoxy” includes O-linked hydrocarbon residues including:

-   -   linear groups of between 1 and 6 atoms (C₁-C₆), or of between 1        and 4 atoms (C₁-C₄). Examples of such alkoxy groups include, but        are not limited to, C₁-methoxy, C₂-ethoxy, C₃-n-propoxy and        C₄-n-butoxy.    -   branched groups of between 3 and 6 atoms (C₃-C₆) or of between 3        and 4 atoms (C₃-C₄). Examples of such alkoxy groups include, but        are not limited to, C₃-iso-propoxy, and C₄-sec-butoxy and        tert-butoxy.

Halo is selected from Cl, F, Br and I. Halo is preferably F.

The term “aryl” includes a single or fused aromatic ring systemcontaining 6 or 10 carbon atoms; wherein, unless otherwise stated, eachoccurrence of aryl may be optionally substituted with up to 5substituents independently selected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy,OH, halo, CN, COOR¹⁴, CF₃ and NR¹⁴R¹⁵; as defined above. Typically, arylwill be optionally substituted with 1, 2 or 3 substituents. Optionalsubstituents are selected from those stated above. Examples of suitablearyl groups include phenyl and naphthyl (each optionally substituted asstated above). Arylene refers the divalent radical derived from an arylgroup, and shall be construed in accordance with the definition above.

The term “heteroaryl” includes a 5, 6, 9 or 10 membered mono- orbi-cyclic aromatic ring, containing 1 or 2 N atoms and, optionally, anNR¹⁴ atom, or one NR¹⁴ atom and an S or an O atom, or one S atom, or oneO atom; wherein, unless otherwise stated, said heteroaryl may beoptionally substituted with 1, 2 or 3 substituents independentlyselected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, OH, halo, CN, COOR¹⁴, CF₃ andNR¹⁴R¹⁵; as defined below. Examples of suitable heteroaryl groupsinclude thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl,thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl,pyrazinyl, indolyl, benzimidazolyl, benzotriazolyl, quinolinyl andisoquinolinyl (optionally substituted as stated above). Heteroarylenerefers the divalent radical derived from heteroaryl, and shall beconstrued in accordance with the definition above.

The term “heterocyclyl” is a C-linked or N-linked 3 to 10 memberednon-aromatic, mono- or bi-cyclic ring, wherein said heterocycloalkylring contains, where possible, 1, 2 or 3 heteroatoms independentlyselected from N, NR¹⁴, S(O)_(q) and O; and said heterocycloalkyl ringoptionally contains, where possible, 1 or 2 double bonds, and isoptionally substituted on carbon with 1 or 2 substituents independentlyselected from (C₁-C₆)alkyl, (C₁-C₆)alkoxy, OH, CN, CF₃, halo, COOR¹⁴,NR¹⁴R¹⁵ and aryl.

In the above definitions R¹⁴ and R¹⁵ are independently selected from Hand (C₁-C₆)alkyl.

When a structural formula is defined with a substituent attached to thecore of the molecule by an unspecified, or “floating” bond, for example,as for the group P³ in the case of formula (C), this definitionencompasses the cases where the unspecified substituent is attached toany of the atoms on the ring in which the floating bond is located,whilst complying with the allowable valence for that atom.

In the case of compounds of the invention which may exist in tautomericforms (i.e. in keto or enol forms), for example the compounds of formula(C) or (H), reference to a particular compound optionally includes allsuch tautomeric forms.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in 25 particularfree from bovine spongiform encephalopathy (BSE). Overall, it ispreferred to culture cells in the total absence of animal-derivedmaterials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

Where a disclaimer is defined above, the disclaimed compounds shouldinclude at least one adsorptive moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 4 show the serum concentration (μM) of compounds 2, 5, 1 & 3either free or adsorbed, over a 24 hour period after intramuscularinjection.

FIG. 5 shows muscle levels (μM) of compounds 24 hours after injection.The x-axis shows the compound number; + indicates adsorption to Al—H,whereas—indicated the absence of Al—H. Each bar represents a singleanimal, with 3 animals per group.

FIG. 6 shows SDS-PAGE of a mixture of MenB antigens. The lanes marked‘std’ show purified antigen standards at three concentrations. Antigenswere formulated with Al—H alone or Al—H to which compound 1 had beenadsorbed. ‘Sn’ shows supernatant after adsorption; ‘Des’ showssupernatants after desorption treatment; ‘TCA’ shows supernatants afterprecipitation.

FIG. 7 shows SBA titers against strain NZ98 using 5CVMB formulated with(a) Al—H alone, (b) Al—H+25 μg compound 1, (c) Al—H+100 μg compound 1,(d) compound 1 alone, or (e) Al—H and MenB outer membrane vesicles.

FIG. 8 shows SBA titers against NZ98 using 5CVMB formulated with (a) noadjuvant, (b) Al—H+OMV, (c) 100 μg compound 2, (d) 25 μg compound2+Al—H, or (e) 100 μg compound 2+Al—H.

FIG. 9 shows SBA titers against NZ98 using 5CVMB formulated with (a) noadjuvant, (b) Al—H and vesicles, (c) 100 μg compound 5, (d) 25 μgcompound 5 (e) 100 μg compound 5+Al—H, or (f) 25 μg compound 5+Al—H.

FIGS. 10 and 11 show the fold-increase, compared to vehicle alone, ofserum cytokines after administration of compound 1 or compound 2. Eachcytokine shows two bars: the left-hand bar shows levels after receivingAl—H-adsorbed compound, whereas the right-hand bar shows levels afterreceiving the compound alone.

FIG. 12 shows structures of compounds 1, 2, 3, 4, 5 and 13.

FIG. 13 shows the concentration (μM) of compound 3 over a period of 5hours after incubation with SIF (♦), buffer () or a plasma/buffermixture (▴).

FIG. 14 shows the degree of desorption (%) of compound 1 over a periodof 5 hours after treatment with potassium phosphate at 10 mM (♦), 100 mM(▪) or 500 mM (▴).

FIG. 15 shows neutralization titers against RSV in mice using RSVF/antigen formulated with (a) Al—H, (b) Al—H and 25 μg of compound 2,(c) Al—H and 25 μg of a different TLR4 agonist, (d) Al—H and aparticulate TLR9 agonist consisting of an immunostimulatoryoligonucleotide complexed with a polycationic oligopeptide, or (c) noadjuvant.

FIG. 16 shows serum levels (μM) over time (hours) of compound 74 after100 μg i.m. injection.

FIG. 17A shows total IgG titers at 6 time points after immunisation withinfluenza virus hemagglutinin adjuvanted with Al—H alone (circles) orAl—H+compound 2 (squares). The time points at 7, 11, 16 and 21 daysafter the first dose, then 7 & 14 days after the second dose.

FIG. 17B shows IgG titers 14 days after the second dose. The left-handpair of bars shows titers where the adjuvant was Al—H alone, whereas theright-hand pair of bars is in the Al—H/cmpd 2 group. In each pair theleft-hand bar shows IgG1 titers and the right-hand bar shows IgG2atiters.

FIG. 18 shows F-specific IgG titers in mice using Al—H with compound 2at the indicated amounts. The data marked as (*) used a particulate TLR9agonist for comparison.

FIGS. 19A-D show IgG titers, after 3 intramuscular injections, againstHla-H35L (FIG. 19A); EsxAB (FIG. 19B); Sta006 (FIG. 19C); and Sta011(FIG. 19D). In each panel the four groups are, from left to right: Al—Hadjuvant alone; Al—H+compound 2; Combo-I+Al—H; Combo-I+Al—H+compound 2.A* indicates a statistically significant difference (p<0.05) againstadjuvant alone. A** indicates a statistically significant difference(p<0.05) against Combo-I+Al—H.

FIG. 20 shows the % of CD40-positive cells. Lines are: *=LPS; X=Al—H andcompound 2, serially diluted; triangle=Al—H and compound 2, with fixedAl+++ dose; and square=soluble compound 2 without Al—H. (A) is anegative control of medium alone. (B) is a negative control of bufferalone when testing soluble compound 2 or Al—H alone when testingadsorbed compound 2. The other values on the X-axis are the amount oftest compound.

FIG. 21 shows the % of CD80-positive cells. The format is the same as inFIG. 20.

FIG. 22 shows chromatograms of Al—H/compound 2 before and afterautoclaving.

FIGS. 23A-D shows serum levels (nM) over time (hours on X-axis) ofcompounds: 6 (FIG. 23A); 67 (FIG. 23B); 68 (FIG. 23C); and 71 (FIG. 23D)after intramuscular injection (100 μg).

FIG. 24 shows muscle levels (nM in quadriceps) of compounds (A) 67 (B)71 and (C) 68 after intramuscular injection of 100 μg compound 24 hoursearlier when adsorbed to Al—H. Arrows show the absence of detectablelevels for unabsorbed compounds.

FIG. 25 shows B cell activation (% of CD 19+ cells which are CD69+) inresponse to compounds 67 (b & e), 68 (c & f) & 71 (d & g) whenadministered with histidine buffer (soluble; b, c & d) or with Al—H(adsorbed; e, f & g). Resiquimod was used as a positive control (h), andbuffer alone as a negative control (a). For all of a-h, the left-handbar shows cells in inguinal lymph nodes and the right-hand bar showscells in the spleen.

FIG. 26 shows gp140-specific IgG titers at day 35 after immunisations ondays 0 & 21. Adjuvants were: (A) unadjuvanted; (B) Al—H; (C) Al—H+a TLR4agonist; (D) compound 2 adsorbed to Al—H; (E) MF59 emulsion.

MODES FOR CARRYING OUT THE INVENTION Phospho-ModifiedBenzonaphthyridines

Compound ‘O’, a useful TLR7 agonist, is prepared as disclosed in example48 of reference 4:

Various modifications to this ‘parent’ benzonaphthyridine compound aremade to improve its physicochemical properties, and in particular toincrease its solubility in water and to confer on it the ability toadsorb to an aluminium hydroxide adjuvant, thereby ensuring in vivodelivery in a controlled manner with prolonged persistence at the siteof injection. With this intent, the compounds referred to above asCompounds 1-15 and 60-62 are synthesized as disclosed in reference 10(see also reference 72).

Example compounds 1, 2, 3, 4, 5, and 13 (FIG. 12) include a phosphonategroup attached to compound O's phenyl ring via ethylene glycol linkers.The phosphonate provides a group which could undergo ligand exchangewith an aluminium salt whereas the ethylene glycol linkers increasewater solubility of the compounds. Compounds 2, 3, 4 & 5 additionallyinclude a carboxyl moiety which also serves to improve water solubility.The phosphonate in compounds 1, 3, 4 and 5 includes two electronegativefluoro substituents on the a carbon, aiming to improve solubility.

Example compound 102 includes a phosphate group attached to compound O'sphenyl ring. The phosphate provides a group which could undergo ligandexchange with an aluminium salt. Compound 102 is synthesised asdisclosed in reference 10.

Adsorption Studies—Aluminium Hydroxide

Adsorption of compounds to aluminium hydroxide (Al—H) is studied atvarious pH.

Compound 13 (0.5 mg/mL) is dissolved in 10 mM NaOH (pH 6.5 or pH 9) andadded to aluminium hydroxide adjuvant (2 mg/mL) resulting in a 100μg/dose formulation. The supernatant is evaluated with HPLC using aballistic gradient (from 10% CH₃CN-0.1% TFA to 100% CH₃CN-0.1% TFA in2.5 minutes) on a C18 (50 cm×4.6 mm) ACE column at 45° C. To evaluatethe effect of supernatant temperature and incubation time on binding,the supernatant is evaluated at room temperature and at 37° C. after 1hour, 5 hours and 24 hours. A control without aluminium hydroxide isalso evaluated. HPLC chromatograms for compound 1 formulations with andwithout aluminium hydroxide, at either temperature, and at either pH,show that compound 1 is not present in the supernatant at any time pointwhen aluminium hydroxide is present, suggesting it has adsorbed to themetal salt.

Compound 12 (1 mg/mL) is tested in the same way at pH 9. Compound 12 isalso tested at pH 6.7 in 10 mM histidine buffer. Again, compound 10 isnot present in the supernatant when aluminium hydroxide is included inthe formulation, at either pH and either temperature.

An organic solvent extraction method is used to evaluate whethercompound 13 is covalently bound to the aluminium hydroxide. Theformulation is prepared as follows: 2 mg/ml aluminium hydroxide, 100μg/dose compound 13, 10 mM histidine buffer; adjust pH to 9. A controlformulation without aluminium hydroxide is also prepared. 1 ml of theformulation is mixed with 1 ml of KH₂PO₄ 1M pH 9 (0.5M final conc, pH 9)and left in gentle agitation overnight at 37° C. If the compound adsorbsto the aluminium salt by ligand exchange then the free phosphate anionswill displace it. Organic extraction is then performed: 1 ml of eachsample is mixed with 1 ml of n-butanol and vortexed.

After the formation of 2 phases, the upper phase (butanol) is recovered,dried with N₂ and resuspended in MeOH/10 mM NaOH. HPLC analysis is runboth for the formulation supernatants and for the butanol-extractedsamples (C18 column; 0-100% B in 2 min; A=0.1% TFA in H2O; B=0.1% TFA inACN). Increased quantities of compound 13 are observed in thesupernatant of the formulation treated with KH₂PO₄, indicatingdisplacement of compound 13 by the phosphate anions i.e. desorption.

Adsorption of compounds 1, 2, 3, 4, 5 (and also of further compounds 6,67, 68 and 71 described below) in 10 mM histidine buffer, pH 6.5, isalso assessed and quantified. These compounds are water-soluble at >1mg/ml and adsorption to Al—H is as follows:

Compound 1 2 3 4 5 6 67 68 71 % 98.2% 97.0% 96.2% 96.0% 94.5% 88% 82%94% 85%

In contrast to the high proportion of adsorption seen with thesephospho-modified compounds, ‘parent’ compound O fails to adsorb to theAl—H adjuvant.

Phospho compound 1 is intrinsically fluorescent. Confocal microscopy ofthe Al—H adjuvant (3 mg/ml) before and after mixing with compound 1(0.25 mg/ml) visually shows that the phospho compound associates withparticles of the insoluble metal salt. Flow cytometry with compound 2shows similar results: compound O is seen as a separate population inthe presence of Al—H, whereas compound 2 co-localizes with the Al—H.

A desorption protocol is used to further confirm binding of thephospho-compounds to Al—H. The compound/Al—H formulation (fluorescent)is treated with 0.5 M phosphate buffer and then washed with either water(for water soluble compounds) or butanol (for poorly water solublecompounds). The washed Al—H is then analyzed and, like Al—H beforehaving been mixed with a phospho-compound, shows no fluorescence.

Stability studies show that the adsorbed compounds are stable forseveral weeks, both in terms of compound stability and adsorption. Allof compounds 1, 2, 3, 4, 5 & 13 show at least 95% adsorption over atleast a 3 week period. Continued study of compounds 2 and 5 show thatthey are stable for 6 weeks or more. Continued RP-HPLC studies ofcompound 2 with Al—H, even in the presence of the 5CVMB antigens (seebelow), showed that the antigens and the TLR7 agonist both remainedstably adsorbed without degradation for at least 24 weeks in aqueousconditions at 4° C., or for at least 4 weeks at 37° C. Osmolarity and pHalso remained within acceptable ranges over the storage period.

FIG. 14 shows desorption of compound 1 stored at 100° C., pH 7, for upto 5 hours. Desorption begins quickly, with increasing levels ofpotassium phosphate (10 mM, 100 mM, 500 mM) leading to increaseddesorption (20%, 60%, 80%) after an hour, but not increasing muchfurther after 5 hours. At 10 mM phosphate the compound remains >70%bound after 5 hours at 100° C., and >80% bound after 5 hours at 70° C.

Various buffer anions were tested for desorption of compound 1, usingboth intrinsic fluorescence and HPLC to follow the compound. Asexpected, KH₂PO₄ (0.5 M, pH 9) was able to completely desorb compound 1from Al—H. A slight reduction in binding (partial desorption) was seenwith glutamate, ascorbate and citrate. In contrast, NaCl and Tris-HCldid not change the adsorption; nor did sodium sulfate or HEPES, orKH₂PO₄ at pH 7.

Compound 102 was tested for adsorption to Al—H. Method: 1. Compound 2Solution: 2 mg of the compound was dissolved in 2 mL water to prepare 1mg/mL solution. Briefly, 2 mL water was added to 2 mg of compound 2 andthe suspension was sonicated for 10 min. 1N NaOH was added in 2 μLincrements until solution went clear; a total of 12 μL of 1N NaOH wasadded. Final pH of solution was determined using a pH strip as ˜10.Solution was further sonicated for 5 min and maintained at roomtemperature. 2. Alum Adsorption: Alum adsorption samples were preparedwith 3 mg/mL aluminum hydroxide and 0.5 mg/mL of compound 2 without abuffer. a) Experimental: 303 μL of water+197 μL of aluminum hydroxide(15.22 mg/mL, lot 1050)+500 μL of 1 mg/mL compound 2, rock at roomtemperature overnight. b) Control: 500 L water+500 μL of 1 mg/mLcompound 2, rock at room temperature overnight. Results: 1) Visual: Uponaddition of compound 2 for experimental vial, the suspension becamewhitish and cloudy, similar to that observed with phosphonates andfluorophosphonates (Compounds 2 and 5). 2) RP-HPLC: Adsorption andrecovery were determined by RP-HPLC. Standards used were 400, 40 and 4μg/mL; Slope was Area—18000× Concentration (μg/mL). a) AdsorptionEfficiency: 10× diluted supernatant was analyzed. i) Experimental:Area=133708; Concentration (1×)—74.3 μg/mL; Efficiency=85.1%. ii)Control: Area=923681; Concentration (1×)=513.2 j±g/mL; Efficiency=−2.6%(equivalent to 0%). b) Recovery: 10× diluted supernatant after boilingwith 0.5M Na₂HPO₄ (pH9). i) Experimental: Area—931578; Concentration(1×)=517.5 μg/mL; Recovery=103.5%. ii) Control: Area=870826;Concentration (1×)=483.8 μg/mL; Recovery=96.8%. Conclusions:Phosphate-containing TLR7 modulator compound 102 adsorbs to aluminumhydroxide at >80% adsorption efficiency to deliver 50 μg dose in 100 μLinjection volume.

Zeta Potential

The surface charge of Al—H particles (2 mg/ml) was measured in theabsence of compound 2, or with increasing concentrations from 10-200 μg.The zeta potential measurement is based on the scattering of light ofparticulate systems. Formulations were prepared in 10 mM histidinebuffer with NaCl, then diluted 1:10 in formulation buffer. Results wereas follows:

Compound 2 (μg) ζ potential (mV) % adsorption 0 +20 ± 2 — 10 +23.5 ± 0.6100 25 +17 ± 1 100 50 +10.6 ± 0.7 100 100 −11.5 ± 0.8 100 150 −28.9 ±0.1 99 200 −41 ± 2 98.8

Thus the net charge on the Al—H particles decreases as the SMIP isadded, inverting polarity between 50 and 100 μg of SMIP. This shows thatthe SMIP is associating with the Al—H particles and modifies thealuminium salt's surface charge.

A net negative charge with 100 μg of SMIP does not exclude thepossibility that the Al—H surface has regions with a partial positivecharge. Such regions may be available for antigen adsorption bycharge-based mechanisms.

Adsorption Studies—Aluminium Phosphate

Adsorption of compounds 1, 2 and 5 to an aluminium phosphate (Al—P)adjuvant is also studied. The Al—P adjuvant is chemically ahydroxyphosphate salt with a PO₄/Al molar ratio of about 0.9 and a PZCabout 5.7. The adjuvant is tested in ‘plain’ form or after pre-treatmentwith increasing concentrations of phosphate buffer (10 mM, 50 mM, 100mM, 250 mM and 500 mM) in an attempt to saturate adsorptive sites on theadjuvant. Incubation was for 8 hours in 10 mM histidine buffer with 0.4mg/ml of the compound and 3 mg/ml Al—P.

Results are as follows, showing % adsorption for two experiments percompound:

Pre-treatment with phosphate buffer (mM) 0 10 50 100 250 500 Cmpd 1 91.684.2 21.4 10.1 17.1 14.6 Cmpd 1 85.3 8.7 10.9 14.5 18.2 Cmpd 2 97.4 92.381.6 61.6 51.9 67.5 Cmpd 2 94.0 81.5 59.2 5.2.0 62.2 Cmpd 5 60 0.4 5.90.0 1.8 0.0 Cmpd 5 0.0 2.4 0.0 2.0 0.0

Thus compounds 2 and 5 adsorb to Al—P with good efficiency (>90%)whereas compound 5 adsorbs at lower efficiency (60%). Pre-treatment ofAl—P with phosphate solution inhibits adsorption of all compounds, andthe degree of adsorption is dependent on the concentration of phosphatein the pre-treatment solution. For compound 5 a 10 mM Pi solution isenough to inhibit completely adsorption, whereas for compound 1 a 50 mMsolution lowers the adsorption to a 20% and for compound 2 a 100 mMsolution lowers the adsorption to ˜60%.

Separate experiments with compound 1 showed that pre-treatment of analuminium phosphate adjuvant with 10 mM potassium phosphate reducedadsorption from 79.8% to 23.5%, and that pre-treatment with 50 mM or 100mM completely inhibited adsorption.

Adsorption Studies—Calcium Phosphate

Adsorption of compound 2 to a commercially-available calcium phosphateadjuvant was studied at pH 6.4. without histidine buffer. Twoformulations are prepared, both with 1.12 mg/ml Ca⁺⁺ but with either0.25 mg/mL or 0.125 mg/ml compound 2. Adsorption was around 90% for bothformulations.

Autoclaving

Compound 2 was adsorbed to Al—H and was analysed by HPLC before andafter autoclaving. FIG. 22 shows that there is no change in thechromatographic profile. Mass spectrometry was also used and there wasalso no change in MS peaks in pre- and post-autoclaved material. Thusthe SMIPs can be sterilised by autoclaving even when adsorbed.

Formula (C)—Adenine Compounds

The following phospho-compound is synthesized, based on the parentadenine compounds disclosed in references 3, 11-17, 21 & 23-25 (inparticular, references 3 & 24):

Compound 6 can be synthesized according to according to the scheme shownbelow, the reference numerals referring to the compounds shown in thescheme below, and not relating to the compound numbering establishedelsewhere in this application. The compound identified with referencenumeral 29 in the scheme below is Compound 6 of the invention.

With reference to the above scheme, alkylation of commercially availableadenine 24 with benzyl bromide 10 (described in ref. 88, Ex 25 Step 1)provides intermediate 25. The aryl chloride of 25 is then substitutedwith 2-methoxyethanol to provide intermediate 26. Bromination of 26 thenfurnishes intermediate 27. Treatment of 27 with sodium methoxide thenprovides 28. Intermediate 28 is then hydrolyzed with bromotrimethylsilane to deliver phosphonic acid 29.

The experimental details of the above synthesis of Compound 6 are asfollows.

Step 1: diethyl4-((6-amino-2-chloro-9H-purin-9-yl)methyl)benzylphosphonate

To a solution of commercially available 2-chloro-9H-purin-6-amine (1equiv.) in DMF (0.50 M) was added potassium carbonate (1.2 equiv.) anddiethyl 4-(bromomethyl)benzylphosphonate (1 equiv.) (described in ref.88, Ex 25 Step 1). The reaction mixture was then heated to 60° C. for 5h. At this point the reaction mixture was allowed to cool to roomtemperature and the volatiles were removed in vacuo. The resultingresidue was purified by a COMBIFLASH™ system (ISCO) using a gradient of0-5% MeOH/DCM to provide the title compound (18%) as a solid.

Step 2: diethyl4-((6-amino-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzylphosphonate

To a solution of diethyl4-((6-amino-2-chloro-9H-purin-9-yl)methyl)benzylphosphonate (1 equiv.)in 2-methoxyethanol (0.10 M) was added sodium hydride (1.3 equiv.). Thereaction mixture was then heated to 120° C. for 18 h. At this point thereaction mixture was allowed to cool to room temperature. The mixturewas then diluted with water and EtOAc. This mixture was transferred to aseparatory funnel and washed with water three times. The organic layerwas then separated, dried over anhydrous Na₂SO₄ and the volatiles wereremoved in vacuo. The resulting residue was purified by a COMBIFLASH™system using a gradient of 0-10% MeOH/DCM to provide the title compoundin quantitative yield as a solid.

Step 3: diethyl4-((6-amino-8-bromo-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzylphosphonate

To a solution of diethyl4-((6-amino-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzylphosphonate(1 equiv.) in acetic acid (0.20 M) was added sodium acetate (15 equiv.)and bromine (13 equiv.). The reaction mixture was then allowed to stirat room temperature for 18 h. At this point the reaction was quenched bythe addition of sodium thiosulfate. The mixture was then diluted withwater and DCM. This mixture was transferred to a separatory funnel andwashed with DCM three times. The combined organic layers were then driedover anhydrous Na₂SO₄ and the volatiles were removed in vacuo. Theresulting residue was purified by a COMBIFLASH™ system using a gradientof 0-5% MeOH/DCM to provide the title compound (14%) as a solid.

Step 4: diethyl4-((6-amino-8-methoxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzylphosphonate

To a solution of diethyl4-((6-amino-8-bromo-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzylphosphonatein methanol (0.10 M) was added sodium methoxide (20 equiv.). Thereaction mixture was then allowed to stir at 70° C. for 18 h. At thispoint the reaction mixture was allowed to cool to room temperature andthen quenched by the addition of ammonium chloride. The mixture was thendiluted with DCM, transferred to a separatory funnel and washed with DCMthree times. The combined organic layers were then dried over anhydrousNa₂SO₄ and the volatiles were removed in vacuo. The resulting solidcompound (38%) was carried onto the next step without furtherpurification.

Step 5:(4-((6-amino-2-(2-methoxyethoxy)-8-oxo-7H-purin-9(8H)-yl)methyl)benzyl)phosphonicacid

To a solution of diethyl4-((6-amino-8-methoxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzylphosphonate(1 equiv.) in CH₂Cl₂ (0.10 M) at 0° C. was slowly added trimethylsilylbromide (10 equiv.). After 1 h the ice-bath was removed and the reactionmixture was allowed to stir at 22° C. for 2 h. At this point thevolatiles were removed in vacuo and the resulting residue was purifiedby Reverse Phase-HPLC using a 20-90% 0.5 mM NH₄OAc (in MeCN) to 10 mMNH₄OAc (in water) gradient to deliver the title compound (29%) as asolid.

¹H NMR (Dimethylsulfoxide-d6): δ 7.16-7.11 (br, 4H), 6.58 (br, 2H), 4.77(s, 2H), 4.25 (t, 2H, J=4.0 Hz), 3.58 (t, 2H, J=4.0 Hz), 3.27 (s, 3H),2.73 (s, 1H), 2.67 (s, 1H). LRMS [M+H]=410.1

The activity of this compound is assessed in an assay using HEK293 cellswhich are stably transfected with human TLR7 and an NF-κB-drivenluciferase reporter vector (pNifty-Luciferase). As a control assay,normal HEK293 transfected with pNifty-Luc are used. Cells are culturedin DMEM supplemented with 2 mM L-glutamine, 10% heart inactivated FBS,1% penicillin and streptomycin, 2 μg/ml puromycin and 5 μg/ml ofblasticidin. Bright-Glo™ Luciferase assay buffer and substrate aresupplied by Promega. Cells are plated at 25,000 cells/well in 384-wellplates in a final volume of 50 μl of media. Cells are allowed to adhereto the plates after overnight (18 hours) culture at 37° C. and 5% CO₂.Serially-diluted experimental and positive control compounds are thendispensed to each well and incubated for 7 hours at 37° C. and 5% CO₂.Cells stimulated with DMSO alone serve as negative controls. After theincubation, 30 μl of the pre-mix assay buffer and substrate buffer areadded to each well according to manufacturer's instructions. Theluminescence signal is read on a CLIPR machine with an integration timeof 20 seconds per plate. Dose response curves are generated for eachcompound and EC₅₀ values were determined as the concentration that gives50% of the maximal signal. EC₅₀ values are also compared to the activityof resiquimod (set to 100%).

Compound 6 shows an EC₅₀ of 0.41 μM or 93%. In splenocytes it shows anEC₅₀ of 0.98 μM (210%). In human PBMCs it shows an EC₅₀ of 1.0 (35%). Itis soluble in histidine buffer at pH 6.8.

Formula (C)—Deazapurine Compounds

The following phospho-compound 67 is synthesized (a deazapurine analogof adenine compound 6), based on the parent deazapurine compoundsdisclosed in references 18-20:

Compound 67 can be synthesized according to according to the schemeshown below, the reference numerals referring to the compounds shown inthe scheme below, and not relating to the compound numbering establishedelsewhere in this application. The compound identified with referencenumeral 16 in the scheme below is Compound 67 of the invention.

With reference to the above scheme, alkylation of pyridine 9 (describedin ref. 18, Preparation 67) with benzyl bromide 10 (described in ref.88, Ex 25 Step 1) furnishes intermediate 11. Amination of 11 withammonia then delivers pyridine 12. The aryl bromide of 12 is thensubstituted with 2-methoxyethanol to provide intermediate 13.Hydrogenation of 13 then furnishes diamine 14. Treatment of 14 withacetic acid under microwave conditions then delivers deazapurine 15.Intermediate 15 is then hydrolyzed with bromotrimethyl silane to deliverphosphonic acid 16.

The experimental details of the above synthesis of Compound 67 are asfollows.

Step 1: ethyl(2,6-dibromo-3-nitropyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate

To a solution of ethyl (2,6-dibromo-3-nitropyridin-4-yl)carbamate (1equiv.) (described in ref. 18, Preparation 67) in acetonitrile (0.40 M)was added triethyl amine (1.5 equiv.) and diethyl4-(bromomethyl)benzylphosphonate (1.3 equiv.) (described in ref. 88, Ex25 Step 1). The reaction mixture was then heated to 60° C. for 18 h. Atthis point the reaction mixture was allowed to cool to room temperatureand the volatiles were removed in vacuo. The resulting residue waspurified by a COMBIFLASH™ system using a gradient of 0-5% MeOH/DCM toprovide the title compound (38%) as a solid.

Step 2: ethyl(2-amino-6-bromo-3-nitropyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate

To a solution of ethyl(2,6-dibromo-3-nitropyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate(1 equiv.) in THF (0.40 M) was added a 7 M solution of ammonia inmethanol (5 equiv.). The reaction mixture was then allowed to stir atroom temperature for 48 h. At this point the volatiles were removed invacuo. The resulting residue was purified by a COMBIFLASH™ system usinga gradient of 0-5% MeOH/DCM to provide the title compound (68%) as asolid.

Step 3: ethyl(2-amino-6-(2-methoxyethoxy)-3-nitropyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate

To a solution of ethyl(2-amino-6-bromo-3-nitropyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate(1 equiv.) in THF (0.30 M) was added sodium hydride (5 equiv.) in asolution of 2-methoxyethanol (0.50 M). The reaction mixture was allowedto stir at room temperature for 2 h. At this point the volatiles wereremoved in vacuo. The resulting residue was purified by a COMBIFLASH™system using a gradient of 0-5% MeOH/DCM to provide the title compound(68%) as a solid.

Step 4: ethyl(2,3-diamino-6-(2-methoxyethoxy)pyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate

To a solution of ethyl(2-amino-6-(2-methoxyethoxy)-3-nitropyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate(1 equiv.) in EtOH (0.05 M) was added 10% Pd/C (50% equiv. by weight) ina Paar Shaker Flask. The reaction mixture was placed in a Paar Shaker at55 psi for 4 h. At this point the reaction mixture was passed through apad of Celite, washing with a 2:1 mixture of CHCl₃:MeOH. The combinedorganic layers were dried over anhydrous Na₂SO₄ and the volatiles wereremoved in vacuo. The resulting residue was used in the next stepwithout further purification.

Step 5: diethyl4-((4-amino-6-(2-methoxyethoxy)-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)methyl)benzylphosphonate

A solution of ethyl(2,3-diamino-6-(2-methoxyethoxy)pyridin-4-yl)(4-((diethoxyphosphoryl)methyl)benzyl)carbamate(1 equiv.) in AcOH (0.15 M) was heated in a microwave at 100° C. for 5min. At this point the volatiles were removed in vacuo. The resultingresidue was purified by a COMBIFLASH™ system using a gradient of 0-10%MeOH/DCM to provide the title compound (63% over two steps) as a solid.

Step 6:(4-((4-amino-6-(2-methoxyethoxy)-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)methyl)benzyl)phosphonicacid

To a solution of diethyl4-((4-amino-6-(2-methoxyethoxy)-2-oxo-2,3-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)methyl)benzylphosphonate(1 equiv.) in CH₂Cl₂ (0.10 M) at 0° C. was slowly added trimethylsilylbromide (10 equiv.). After 1 h the ice-bath was removed and the reactionmixture was allowed to stir at 22° C. for 18 h. At this point thevolatiles were removed in vacuo and the resulting residue was purifiedby Reverse Phase-HPLC using a 20-90° %. 0.5 mM NH₄OAc (in MeCN) to 10 mMNH₄OAc (in water) gradient to deliver the title compound (41%) as asolid.

¹H NMR (Dimethylsulfoxide-d6): δ 7.09-7.05 (br, 4H), 5.79 (s, 1H), 5.63(br, 2H), 4.78 (s, 2H), 4.17 (t, 2H, J=4.8 Hz), 3.55 (t, 2H, J=4.8 Hz),3.23 (s, 3H), 2.71 (s, 1H), 2.66 (s, 1H). LRMS [M+H]=409.1

Compound 67 shows an EC₅₀ of 6.5 μM or 103% in HEK293 cells. Insplenocytes it shows an EC₅₀ of 9.3 μM (142%). In human PBMCs it showsan EC₅-of 0.8 (59%). It is soluble in histidine buffer at pH 6.8.

Formula (D)

The following phospho-compound is synthesized, based on the parentsubstituted imidazoquinoline compounds disclosed in references 2 & 22:

Compound 7 has a EC₅₀ of 66 μM (55%) in the HEK293 assay disclosed abovebut is inactive in splenocytes and hPBMCs. It is insoluble in histidinebuffer at pH 6.8.

Modified forms of compound 7, with longer ethylene glycol linkers, arealso prepared (compounds 37 and 68). Compound 68 has a EC₅₀ of 28M (75%)in HEK293 cells but, again, is inactive in splenocytes and hPBMCs.Unlike compound (7) it is soluble in histidine buffer at pH 6.8.

Compound 68 can be synthesized according to according to the schemeshown below, the reference numerals referring to the compounds shown inthe scheme below, and not relating to the compound numbering establishedelsewhere in this application. The compound identified with referencenumeral 23 in the scheme below is Compound 68 of the invention.

With reference to the above scheme, commercially available quinoline 17is substituted with amine 18 to deliver intermediate 19. Reduction ofthe nitro group in 19 then provides intermediate 20. Coupling ofintermediate 20 with 2-ethyoxyacetic acid then furnishes intermediate21. Amination of intermediate 21 with ammonia then affordsimidazoquinoline 22. Hydrolysis of 22 with bromotrimethyl silane thenfurnishes phosphonic acid 23.

The experimental details of the above synthesis of Compound 68 are asfollows.

Step 1: diethyl(2-(2-(2-(2-((2-chloro-3-nitroquinolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)phosphonate

To a solution of commercially available 2,4-dichloro-3-nitroquinoline (1equiv.) in triethyl amine (0.30 M) was added commercially availablediethyl (2-(2-(2-(2-aminoethoxy)ethoxy) ethoxy)ethyl)phosphonate (1.3equiv.) (provided by PHARMARON). The resulting reaction mixture wasstirred at 70° C. for 2 h. At this point the volatiles were removed invacuo. The resulting residue was purified by a COMBIFLASH™ system usinga gradient of 0-5% MeOH/DCM to provide the title compound (63%) as asolid.

Step 2: diethyl(2-(2-(2-(2-((3-amino-2-chloroquinolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)phosphonate

To a solution of diethyl(2-(2-(2-(2-((2-chloro-3-nitroquinolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)phosphonate(1 equiv.) in EtOAc (0.10 M) was added 10% Pt/C (5% equiv. by weight)and MgSO₄ (2 equiv.) in a Paar Shaker Flask. The reaction mixture wasplaced in a Paar Shaker at 40 psi for 7 h. At this point the reactionmixture was passed through a pad of Celite, washing with a 2:1 mixtureof CHCl₃:MeOH. The combined organic layers were dried over anhydrousNa₂SO₄ and the volatiles were removed in vacuo. The resulting residuewas purified by a COMBIFLASH™ system using a gradient of 0-10% MeOH/DCMto provide the title compound (97%) as a solid.

Step 3: diethyl(2-(2-(2-(2-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)phosphonate

To a solution of diethyl(2-(2-(2-(2-((3-amino-2-chloroquinolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)phosphonate(1 equiv.) in DMF (0.25 M) was added(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (1 equiv.) and 2-ethoxyacetic acid 21 (1.2 equiv.).The resulting reaction mixture was heated to 50° C. for 18 h. At thispoint the reaction mixture was allowed to cool to room temperature. Themixture was then diluted with water and EtOAc. This mixture wastransferred to a separatory funnel and washed with water three times.The organic layer was then separated, dried over anhydrous Na₂SO₄ andthe volatiles were removed in vacuo. The resulting residue was purifiedby a COMBIFLASH™ system using a gradient of 0-10% MeOH/DCM to providethe coupled intermediate (19%) as a solid. A solution of thisintermediate (I equiv.) in 2 M ammonia in isopropanol (0.06 M) was thenheated at 100° C. for three days. At this point the volatiles wereremoved in vacuo. The resulting residue was purified by a COMBIFLASHsystem using a gradient of 0-10% MeOH/DCM to provide the title compound(62%) as a solid.

Step 4:(2-(2-(2-(2-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)phosphonicacid

To a solution of diethyl(2-(2-(2-(2-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)phosphonate(1 equiv.) in CH₂Cl₂ (0.10 M) at 0° C. was slowly added trimethylsilylbromide (10 equiv.). After 1 h the ice-bath was removed and the reactionmixture was allowed to stir at 22° C. for 18 h. At this point thevolatiles were removed in vacuo and the resulting residue was purifiedby Reverse Phase-HPLC using a 20-90% 0.5 mM NH₄OAc (in MeCN) to 10 mMNH₄OAc (in water) gradient to deliver the title compound (26%) as asolid.

¹H NMR (CDCl₃): δ 8.00-7.76 (m, 2H), 7.51-7.28 (m, 2H), 4.91-4.72 (m,4H), 3.98-3.84 (m, 2H), 3.72-3.54 (m, 4H), 3.48-3.38 (m, 2H), 3.30-3.29(m, 2H), 3.13-3.00 (m, 2H), 2.10-1.96 (m, 2H), 1.31-1.11 (m, 3H). LRMS[M+H]=483.1.

Formula (E)

The following phospho-compound is synthesized, based on the parentpyrimidine compounds disclosed in reference 5 e.g. the ester in example21:

Compound 8 has a EC₅₀ of 7.3 μM (81%) in the HEK293 assay disclosedabove. In splenocytes it shows an EC₅₀ of 3.1 μM (184%). In hPBMCs itshows an EC₅₀ of 10.4 (50%). It is insoluble in histidine buffer at pH6.8.

Further compounds of formula (E) are prepared:

Compound (69) shows an EC₅₀ of 47.4 μM (36%) in HEK293 cells but isinactive in splenocytes and hPBMCs. It is insoluble in histidine bufferat pH 6.8. Compound 70 also had poor solubility.

Compound 71 shows an EC₅₀ of 2.7 μM (53%) in HEK293. In splenocytes itshows an EC₅₀ of 10.5 μM (239%). In hPBMCs it shows an EC₅₀ of 9.5(50%). It is soluble in histidine buffer at pH 6.8.

Compound 71 can be synthesized according to according to the schemeshown below, the reference numerals referring to the compounds shown inthe scheme below, and not relating to the compound numbering establishedelsewhere in this application. The compound identified with referencenumeral 8 in the scheme below is Compound 71 of the invention.

With reference to the above scheme, commercially available phenol 1 isalkylated with bromide 2 (described in ref. 88, Ex 7 Step 1) to deliverintermediate 3. Condensation of intermediate 3 with ethyl acetoacetatethen provides enoate 4. Cyclization of 4 with guanidine carbonate thenprovides pyrimidine 5. Intermediate 5 is then converted to sulfone 6which is substituted with as amylamine do deliver pyrimidine 7.Hydrolysis of 7 with bromotrimethyl silane then furnishes phosphonicacid 8.

Step 1: diethyl(1,1-difluoro-3-(2-(4-formyl-3-methoxyphenoxy)ethoxy)propyl)phosphonate

To a solution of 4-hydroxy-2-methoxybenzaldehyde (1 equiv.) in DMF (0.25M) was added cesium carbonate (2 equiv.). sodium iodide (0.20 equiv.)and diethyl (3-(2-bromoethoxy)-1,1-difluoropropyl)phosphonate (1.2equiv.) (described in ref. 88, Ex 7 Step 1). The reaction mixture wasstirred at 100° C. for 3 h, after which it was allowed to cool to roomtemperature. The resulting mixture was then diluted with water andEtOAc. This mixture was transferred to a separatory funnel and washedwith water three times. The organic layer was then separated, dried overanhydrous Na₂SO₄ and the volatiles were removed in vacuo. The resultingresidue was purified by a COMBIFLASH™ system using a gradient of 0-5%MeOH/DCM to provide the title compound (79%) as a solid.

Step 2: (E)-ethyl2-(4-(2-(3-(diethoxyphosphoryl)-3,3-difluoropropoxy)ethoxy)-2-methoxybenzylidene)-3-oxobutanoate

To a solution of diethyl(1,1-difluoro-3-(2-(4-formyl-3-methoxyphenoxy)ethoxy)propyl)phosphonate(1 equiv.) in toluene (0.50 M) was added piperidine (0.10 equiv.),acetic acid (0.50 equiv.) and ethyl acetoacetate (0.2 equiv.). Thereaction mixture was then heated to 110° C. for 18 h. At this pointadditional piperidine (0.10 equiv.), acetic acid (0.50 equiv.) and ethylacetoacetate (0.50 equiv.) were added to the reaction mixture andheating at 110° C. was continued for 4 h. At this point the reactionmixture was allowed to cool to room temperature and the volatiles wereremoved in vacuo. The resulting residue was purified by a COMBIFLASH™system using a gradient of 0-5% MeOH/DCM to provide the title compound(78%) as a solid.

Step 3: diethyl(3-(2-(4-((2-amino-4-hydroxy-6-methylpyrimidin-5-yl)methyl)-3-methoxyphenoxy)ethoxy)-1,1-difluoropropyl)phosphonate

To a solution of (E)-ethyl2-(4-(2-(3-(diethoxyphosphoryl)-3,3-difluoropropoxy)ethoxy)-2-methoxybenzylidene)-3-oxobutanoate(1 equiv.) in MeOH (0.20 M) was added guanidine carbonate (1.1 equiv.)and the reaction mixture was heated to 80° C. for 3 h. At this point thereaction mixture was allowed to cool to room temperature and it was thendiluted with water and EtOAc. The resulting mixture was transferred to aseparatory funnel and washed with EtOAc three times. The combinedorganic layers were dried over anhydrous Na₂SO₄ and the volatiles wereremoved in vacuo. The resulting residue was purified by a COMBIFLASH™system using a gradient of 0-10% MeOH/DCM to provide the title compound(15%) as a solid.

Step 4:2-amino-5-(4-(2-(3-(diethoxyphosphoryl)-3,3-difluoropropoxy)ethoxy)-2-methoxybenzyl)-6-methylpyrimidin-4-yl2,4,6-trimethylbenzenesulfonate

To a solution of diethyl(3-(2-(4-((2-amino-4-hydroxy-6-methylpyrimidin-5-yl)methyl)-3-methoxyphenoxy)ethoxy)-1,1-difluoropropyl)phosphonate(1 equiv.) in THF (0.40 M) was added 1,4-diazabicyclo[2.2.2]octane (1.8equiv.) and 2,4,6-trimethylbenzene-1-sulfonyl chloride (1.3 equiv.). Thereaction mixture was allowed to stir at room temperature for 18 h afterwhich it was quenched with aqueous HCl (0.10 N). The resulting mixturewas transferred to a separatory funnel and washed with chloroform threetimes. The combined organic layers were dried over anhydrous Na₂SO₄ andthe volatiles were removed in vacuo. The resulting residue was purifiedby a COMBIFLASH™ system using a gradient of 0-5% MeOH/DCM to provide thetitle compound (41%) as a solid.

Step 5: diethyl(3-(2-(4-((2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl)methyl)-3-methoxyphenoxy)ethoxy)-1,1-difluoropropyl)phosphonate

To a solution of2-amino-5-(4-(2-(3-(diethoxyphosphoryl)-3,3-difluoropropoxy)ethoxy)-2-methoxybenzyl)-6-methylpyrimidin-4-yl2,4,6-trimethylbenzenesulfonate (1 equiv.) in EtOAc (0.20 M) was addedTFA (1.1 equiv.) and amylamine (3.3 equiv.). The reaction mixture wasthen heated to 100° C. for 18 h. At this point the reaction mixture wasallowed to cool to room temperature and the volatiles were removed invacuo. The resulting residue was purified by a COMBIFLASH™ system usinga gradient of 0-10% MeOH/DCM to provide the title compound (76%) as asolid.

Step 6:(3-(2-(4-((2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl)methyl)-3-methoxyphenoxy)ethoxy)-1,1-difluoropropyl)phosphonicacid

To a solution of diethyl(3-(2-(4-((2-amino-4-methyl-6-(pentylamino)pyrimidin-5-yl)methyl)-3-methoxyphenoxy)ethoxy)-1,1-difluoropropyl)phosphonate(1 equiv.) in CH₂Cl₂ (0.10 M) at 0° C. was slowly added trimethylsilylbromide (10 equiv.). After 1 h the ice-bath was removed and the reactionmixture was allowed to stir at 22° C. for 18 h. At this point thevolatiles were removed in vacuo and the resulting residue was purifiedby Reverse Phase-HPLC using a 20-90% 0.5 mM NH₄OAc (in MeCN) to 10 mMNH₄OAc (in water) gradient to deliver the title compound (70%) as asolid.

¹H NMR (Methanol-d4): δ 6.81 (d, 1H, J=8.4 Hz), 6.62 (s, 1H), 6.48 (d,1H, J=8.4 Hz), 4.09 (t, 2H, J=4.4 Hz), 3.90 (s, 3H), 3.86 (t, 2H, J=7.6Hz), 3.79 (t, 2H, J=4.4 Hz), 3.67 (s, 2H), 3.43 (t, 2H, J=7.2 Hz), 2.30(s, 3H), 2.22 (t, 2H, J=7.6 Hz), 2.06-2.01 (m, 2H), 1.33-1.10 (m, 4H),0.88 (t, 3H, J=7.6 Hz). LRMS [M+H]=533.3.

Formula (III)

The starting compound for preparing compound 19 was(5R,9R)-9-(dodecanoyloxy)-1-(9H-fluoren-9-yl)-3,12-dioxo-2,11-dioxa-7-thia-4-azatricosane-5-carboxylicacid (“TLR2-pre”), prepared as follows:

Step 1: (R)-tert-butyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-mercaptopropanoate (b)

A solution of (N-Fmoc-Cys-OtBu)₂ (7, 1 eq), NEt₃ (3 eq) and DTE(1,4-Dithioerythritol, 2.5 eq) in DCM (0.1 M) was stirred at roomtemperature until complete reduction (1.5 hours). The reaction mixturewas diluted in DCM, washed three times with 5% citric acid, twice withwater, and once with brine. The organic layer was dried over anhydrousNa₂SO₄ and concentrated en vaccuo. The resulting crude was purified byflash chromatography on a COMBIFLASH® system (ISCO) using 0-30%,EtOAc/Hex to give the title product as colorless viscous oil.

Step 2: (R)-tert-butyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-((R)-2,3-dihydroxypropylthio)propanoate(10)

A solution of (2S)-(+)-glycidyl-4-nitrobenzoate (9, 1.1 eq) and 1 M NaOH(1.1 eq) in tBuOH (0.1 M) was stirred at room temperature until completehydrolysis of the nitrobenzoate (30 minutes). To the resulting mixture,a solution of (R)-tert-butyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-mercaptopropanoate (8, 1eq) in tBuOH (1 M) was introduced. The reaction was stirred at roomtemperature for 15 hours. The reaction mixture was concentrated envaccuo to remove tBuOH and dissolved in EtOAc. The EtOAc solution waswashed three times with water, and once with brine.

The resulting crude was purified by flash chromatography on aCOMBIFLASH™ system (ISCO) using 0-90% EtOAc/Hex to give the titleproduct as colorless viscous oil.

Step 3:(R)-3-((R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-tert-butoxy-3-oxopropylthio)propane-1,2-diyldidodecanoate (11)

A solution of (R)-tert-butyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-((R)-2,3-dihydroxypropylthio)propanoate(10, 1 eq) in DCM (0.1 M) was cooled in an ice bath. Pyridine (3.7 eq)was added followed by dodecanoyl chloride (3.7 eq). The reaction mixturewas stirred for 10 minutes then warmed up to room temperature, andstirred for 2 hours. The reaction mixture was diluted with DCM, washedwith saturated aqueous NH₄Cl. The aqueous phase was back extracted withDCM. The combined organic phases were washed with H₂O, and the aqueousphase was back extracted with DCM. The combined organic phases werewashed with brine, dried over anhydrous Na₂SO₄ and concentrated envaccuo. The resulting crude was purified by flash chromatography on aCOMBIFLASH™ system (ISCO) using 0-30% EtOAc/Hex to give the titleproduct as a white solid.

Step 4:(5R,9R)-9-(dodecanoyloxy)-1-(9H-fluoren-9-yl)-3,12-dioxo-2,11-dioxa-7-thia-4-azatricosane-5-carboxylicacid (“TLR-pre”)

A solution of(R)-3-((R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-tert-butoxy-3-oxopropylthio)propane-1,2-diyl didodecanoate (11) in 40% TFA in DCM (0.3 M) wasstirred at room temperature until complete deprotection of tert-butylgroup (2 hr). The reaction mixture was diluted in MTBE, washed threetimes with 1M citric acid (adjusted to pH3), and once with 1:2 1NHCl/brine. The organic layer was dried over anhydrous Na₂SO₄ andconcentrated en vaccuo. The resulting waxy solid was used withoutfurther purification.

This material was used for preparing compounds 17, 19 and 22. Forinstance, compound 19 was prepared as follows:

Step 1: diethyl 2-(2-2-iodoethoxy)ethoxy)ethylphosphonate

1,2-bis(2-iodoethoxy)ethane (1.0 eq) was mixed with triethyl phosphate(1 eq) then heated to 160° C. for 20 minutes by microwave. The crudemixture was purified by flash chromatography on a COMBIFLASH™ systemusing 85-100% EtOAc/Hex to give diethyl2-(2-(2-iodoethoxy)ethoxy)ethylphosphonate as a colorless oil.

Step 2: diethyl 2-(2-(2-azidoethoxy)ethoxyl)ethylphosphonate

To a solution of diethyl 2-(2-(2-iodoethoxy)ethoxy)ethylphosphonate (1eq) in EtOH (0.2 M) was added sodium azide (5 eq) in water (1.4 M). Thereaction mixture was heated at reflux overnight. The mixture was thendiluted with water, extracted with EtOAc (3 times). The combined organiclayers were washed brine, dried over anhydrous Na₂SO₄, and concentrateden vaccuo. The crude material was purified by flash chromatography on aCOMBIFLASH™ system using 0-5% MeOH/DCM to give diethyl2-(2-(2-azidoethoxy)ethoxy)ethylphosphonate as colorless oil.

Step 3: diethyl 2-(2-(2-aminoethoxy)ethoxy)ethylphosphonate

Diethyl 2-(2-(2-azidoethoxy)ethoxy)ethylphosphonate (1 eq) was dissolvedin EtOH (0.1 M). Pd(OH)₂ (0.05 eq) was added to the reaction. Hydrogengas was introduced via a balloon; and the reaction was stirred for 2hours at room temperature. The mixture was filtered through Celite andwashed with MeOH. The solvent was removed en vaccuo and the crudematerial was purified by flash chromatography on a COMBIFLASH™ systemusing 0-10% MeOH/DCM with 0.5% NH₃ to give diethyl2-(2-(2-aminoethoxy)ethoxy)ethylphosphonate as a colorless oil.

Step 4:(1R,1R)-1-(((9H-fluoren-9-yl)methoxy)carbonylamino)-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-13-thia-9-azanonacosylphosphoryldiethyl ester

To a solution of TLR2-pre (1 eq) in DCM (0.1 M) was added diethyl2-(2-(2-aminoethoxy) ethoxy)ethylphosphonate (1.3 eq), DIEA (2.5 eq) andHBTU (1.2 eq). The reaction was stirred at room temperature for 2 hours.The crude mixture was purified by flash chromatography by flashchromatography on a COMBIFLASH™ system using 70-100% EtOAc/Hex to give(11R,15R)-11-(((9H-fluoren-9-yl)methoxy)carbonylamino)-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-13-thia-9-azanonacosylphosphoryldiethyl ester.

Step 5:(11R,15R)-1-amino-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-3-thia-9-azanonacosylphosphoryl diethyl ester

To a solution of(11R,15R)-11-(((9H-fluoren-9-yl)methoxy)carbonylamino)-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-13-thia-9-azanonacosylphosphoryldiethyl ester (1 eq) was added 20% piperidine (50 eq) in acetonitrile.The resulting mixture was diluted with DCM and sonicated for 3 minutes.To the mixture was added to toluene and then concentrated en vaccuo. Thecrude mixture was purified by flash chromatography on a COMBIFLASH™system using 100% EA followed by 0-10% MeOH in DCM to give(11R,15R)-11-amino-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-13-thia-9-azanonacosylphosphoryl diethyl ester.

Step 6:(11R,15R)-11-amino-15-(dodecanoyloxy)-10,18-dioxo-3,6,7-trioxa-13-thia-9-azanonacosylphosphonic acid

To a solution of(11R,15R)-11-amino-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-13-thia-9-azanonacosylphosphoryl diethyl ester (1 eq) in DCM (0.1 M) was added trimethylsilylbromide (10 eq). The reaction mixture was stirred at room temperatureovernight and concentrated. The crude mixture was purified by reversephase high performance liquid chromatography (HPLC) with C4 columneluting with a gradient of 40-100% MeCN/10 mM NH₄OAc (95:5) in 10 mMNH₄OAc (pH 9) to give(11R,15R)-11-amino-15-(dodecanoyloxy)-10,18-dioxo-3,6,17-trioxa-13-thia-9-azanonacosylphosphonic acid as a white solid.

Compounds 17, 19 and 22 were tested for binding to Al—H in 100 mMhistidine buffer at pH 6.5. Binding to Al—H was >80% in all three cases.Further compounds which are useful for adsorbing to Al—H are the “TLR2p”compounds mentioned above. The interaction of four of these (compounds63-66) with Al—H was tested in the same way as for compounds 17, 19 and22; binding was again >80% in all cases.

Impact of Adsorption on Behaviour of Immunopotentiators Cellular Uptake

The autofluorescence of compound 1 was exploited to follow its uptake byhuman PBMCs after adsorption to Al—H. FACS was used to monitor uptake.

Various concentrations of compound 1 were incubated with cells overnightat 37° C. with or without Al—H The presence of Al—H, to which compound 1is adsorbed, produced a large shift in the FACS pattern, with a doseresponse, and increased fluorescence was detected specifically inmonocytes. Thus the Al—H particles can enhance the cellular uptake ofadsorbed compound 1 in comparison to the free compound in solution.Moreover, the results show that the adsorbed compound is retained forlonger in the cells after entry.

The same uptake has been seen in other cell types too.

Pre-treatment of cells with cytochalasin D resulted in a lowerfluorescence intensity associated with compound 2, compared to untreatedcells, indicating that the process of interaction and internalization ofthe adsorbed compound is actively mediated by monocytes.

Addition of trypan blue is able to “quench” compound 2's fluorescence,but trypan blue is not able to permeate live cells. Cells which were notpre-treated with cytochalasin D remained highly fluorescent aftertreatment with trypan blue, meaning that compound 2 is located insidethe cells. Cellular uptake was time-dependent.

Monocyte activation markers CD40 and CD80 were studied in response tocompounds 1, 2 and 5, either alone or adsorbed to Al—H, or to LPS(positive control). The adsorbed compounds were tested at 5 differentconcentrations, either at a fixed Al⁺⁺⁺ dose or with simple dilution.FIG. 20 shows CD40 activation and FIG. 21 shows CD80 activation usingcompound 2; both markers indicate greater activation in the presence ofadsorbed SMIP than with soluble SMIP. The same trend was seen withcompounds 1 and 5.

The cytokine activation profile of compound 2 was compared in solubleand adsorbed form. There was no significant observable difference inIL-8, IL-6, TNF-α or IL-1β responses.

Thus adsorption of compound 2 to Al—H results in an enhanced uptake ofthe SMIP into the cell, and in a higher degree of cell activation whencompared to incubation with unabsorbed SMIP. Cellular uptake of SMIP ismediated by an active process of internalization that is time- anddose-dependent.

Systemic Exposure after In Vivo Delivery

Compounds 1, 2, 3 and 5 are administered to Balb/C mice by intramuscularinjection at 100 μg (4 mg/kg), either with buffer alone or afteradsorption to an Al—H adjuvant. Systemic serum exposure of the compoundsis followed for 24 hours. As shown in FIGS. 1 to 4, whereas theunabsorbed compounds have a high initial serum concentration whichrapidly declines, adsorbed compounds show a much flatter response whichis sustained for a longer period. Similar systemic serum exposureprofiles were seen in rats.

Despite the longer period of exposure, for these compounds the overallsystemic exposure can be reduced by adsorption, as separately measuredby AUC for compounds 2 and 5:

2 5 Compound 8846 9834 +Al—H 4553 13908

Similarly to FIGS. 1-4, FIG. 23 shows serum exposure of compounds (A) 6(B) 67 (C) 68 and (D) 71 for 24 hours after injection. AUCs were asfollows (nM-hr), and for these four compounds were in all cases higherwhen adsorbed:

6 67 68 71 Compound 7176 3573 1391 2093 +Al—H 13424 8110 2759 4219

FIG. 16 shows similar data for a TLR2 agonist (compound 74). AUCs ofthis compound and compound 80, both TLR2 agonists of interest, werereduced by adsorption to Al—H:

74 80 Compound 37425 20749 +Al—H 3389 7264

Cmax values (nM) were as follows, in general showing a reduction whenadsorbed:

TLR7 TLR2 2 5 6 67 68 71 74 80 Compound 5600 9273 6124 4057 2122 26031937 2823 +Al—H 465 3319 5521 4514 1721 1807 317 180

Serum cytokines are measured 24 hours after immunization with compound1, with or without Al—H, or with buffer alone. Levels of IL-6 and mKCare both about ˜4-fold higher after administration of compound 1 withoutAl—H, and levels of MCP-1 are ˜20-fold higher (compared to vehiclealone). In contrast, when administered in combination with Al—H thelevels are increased to a lesser degree, being <2-fold higher (see FIG.10; see also FIG. 11 for results with compound 2). For compounds 67, 68& 71 systemic cytokines were generally reduced or unaffected byadsorption to Al—H, whereas for compound 6 levels of several cytokines(e.g. IFN-γ, IL1-β, IL12-p40) were enhanced.

Adsorption of compound 1 to Al—H decreases both the proportion of CD4+T-cells which are also CD69+ and the proportion of CD19+ B-cells whichare also CD86+, and this effect is seen in both the spleen and indraining lymph nodes. For example, adsorption reduces the proportion ofCD86+ B-cells from ˜75% to ˜15%. A similar reduction in activation of Bcells is seen when compounds 6, 67 & 68 are adsorbed to Al—H (FIG. 25e.g. compare group f to group c). Thus for all tested TLR7 agonistsadsorption to Al—H reduces general activation of B cells.

Muscle levels of compounds 1, 2, 3, 4, 5 & 13 are measured 24 hoursafter intramuscular injection (1001 g) in Balb/C mice (3 per group) incombination with protein antigens, with or without Al—H.

Except for compound 13, the phospho-compounds are undetectable ifinjected without Al—H, but are readily detected if injected with Al—H(see FIG. 5). Compound 13 is poorly soluble in histidine buffer, pH 6.8,which explains its different behaviour. The soluble ‘parent’ compounds,without modifications to favour adsorption, are also cleared from musclevery rapidly.

Results from similar experiments with compounds 67, 68 and 71 are shownin FIG. 24; again, the compounds are undetectable 24 hours afterinjection in free form (arrows), but are readily detected if injectedwith Al—H. Similar results were obtained for compound 6, which again wasundetectable in muscle after 24 hours, and also could not be detected ininguinal lymph nodes or in liver. Levels of compound in muscle 24 hoursafter injection for various compounds were as follows (nM):

TLR7 TLR2 2 5 6 67 68 71 22 72 74 80 Compound 0 0 0 0 0 0 0 0 27843 5072+Al—H 20393 4214 2773 2876 502 924 25344 14484 49027 147151

Thus for all tested compounds adsorption to Al—H retains higher levelsof the soluble phosphonates at the local injection sites.

In summary, adsorption of these SMIPs to Al—H modifies their local andsystemic post-delivery behaviour in vivo. Adsorption has been shown to:(i) increase cellular uptake of SMIPs; (ii) increase residence time ofSMIPs at sites of intramuscular injection, where they can continue toexert an immunostimulatory effect; (iii) reduce levels of general B cellactivation, thus advantageously minimising general and non-specificimmunostimulation; (iv) decrease Cmax; and (v) modify serum exposureprofiles, with the potential either to increase or decrease overallsystemic exposure thus achieving useful in vivo properties as desirede.g. for widespread prophylactic immunisations, adsorption can be usedto reduce systemic exposure and cytokine stimulation, or for emergencyimmunotherapeutic situations it can be used to increase systemicexposure. The use of adsorption to modify the in vivo behaviour of SMIPsin this way has not previously been reported.

Model of In Vivo Desorption

It is known that antigens can desorb from aluminium salts after beingexposed to interstitial or lymph fluids [89, 90]. Al—H-adsorbedphosphonate compounds were incubated at 37° C. with a histidine buffer,a mixture of plasma and a histidine buffer, or with a simulatedinterstitial fluid (SIF) and adsorption was followed for 5 hours. FIG.13 shows an example desorption profile (compound 3). For six testedcompounds the proportion of desorbed antigen after 5 hours was asfollows:

Compound SIF Plasma/His buff His buff 13*  4%  9% 2% 1  90% 43% 2% 3100% 49% 5% 2  84% 59% 3% 5 100% 73% 4% 4 100% 81% 4% *Results withcompound 13 were unreliable.

In histidine buffer alone, all phosphonates exhibit strong binding toAl—H, with only 2-6% being unbound after 5 hours. In plasma, however,the phosphonates quickly dissociate from Al—H, and dissociation was evenmore rapid in SIF. Moreover, in SIF the rate of dissociationqualitatively correlates with the systemic exposure of a compound asseen after intramuscular injection. Thus, for example, compounds 3 and 4had the highest serum exposure and fastest dissociation in SIF.

Toxicology

There were no concerns following repeat-dose local tolerability andtoxicity study in male rats for compound 2 adsorbed to Al—H. Theformulation was not associated with body weight loss, elevation in bodytemperature or adverse clinical observations.

Effect of Adsorption on Immunogenicity—Meningococcus B

Reference 40 discloses a vaccine for serogroup B meningococcus (‘MenB’)made from three separate polypeptides (‘5CVMB’). These polypeptides canadsorb to Al—H, and SDS-PAGE is used to check if this adsorption canstill occur after adsorption of compound 1 to the Al—H.

Compound 1 is dissolved in 10 mM NaOH at 0.5 mg/ml final concentration,then combined with excess Al—H at a 1:6 weight ratio in the presence of10 mM histidine (final concentration). The pH is adjusted to 9.2 and themixture is gently agitated for 3 hours at room temperature, allowing thereaction to occur. The mixture was centrifuged at 5000 g for 10 minutesand the supernatant discarded. The pellet (Al—H with adsorbedcompound 1) is resuspended in the initial buffer to obtain the startingconcentration of Al—H. The pH was adjusted to 6.5. The modified Al—H isthen used for the formulation with the MenB antigens. For comparison,antigens are also formulated in parallel with ‘plain’ Al—H.

The formulated antigens are centrifuged and the supernatant is tested bySDS-PAGE for presence of the three polypeptides. In further tests,supernatants are treated with trichloroacetic acid (TCA) to precipitateproteins. In further tests, formulated antigens are treated with 0.5 Mphosphate buffer prior to analysis, to desorb any adsorbed antigens.FIG. 6 shows SDS-PAGE of supernatants. Pre-adsorption of compound to theAl—H does not prevent adsorption of the MenB antigens. Further studiesshowed that antigen adsorption still occurred even with a 5-fold excessof TLR agonist.

Similar tests are performed with compounds 3 and 4. HPLC analysis showsthat neither compound is observed in the supernatant after mixing withaluminium hydroxide, but they are recovered after desorption treatmentwith 0.5M KH₂PO₄. SDS-PAGE analysis of MenB antigen binding to Al—Hafter pre-adsorption with compound 3 or 4 again shows that the antigensare completely adsorbed.

The MenB antigens are tested for in vivo immunogenic potency using aserum bactericidal antibody (SBA) assay. FIG. 7 shows bactericidaltiters against strain NZ98 of sera obtained after immunization with5CVMB combined with (a) Al—H alone, (b) Al—H+25 μg compound 1, (c)Al—H+100 μg compound 1, (d) compound 1 alone, or (e) Al—H and MenB outermembrane vesicles. Pre-adsorption of compound 1 to Al—H gives a largeincrease in SBA titer, and much greater than would be expected based onresults seen with compound 1 alone. Thus, although general stimulationof B cells is reduced by adsorption (see above), MenB-specific antibodyresponses are enhanced.

Similar effects are seen with other phospho-modified compounds of theinvention. For instance, FIG. 8 shows results with compound 2, and FIG.9 shows results with compound 5 (both sera tested against strain NZ98).These two compounds are selected for further evaluation.

Compound 2 is pre-adsorbed to Al—H to investigate strain coverage ofsera obtained after immunization with a modified 5CVMB in which theGNA2091/1870 fusion protein is replaced by the ‘936-10A-10A’ proteindisclosed in ref. 91 (SEQ ID NO: 126 therein; SEQ ID NO: 4 herein).

The following table shows titers against five different strains afterformulating the three polypeptides with (a) Al—H alone, (b) Al—H+25 μgouter membrane vesicles, (c) Al—H with 100 μg of compound 2, (d) Al—Hwith 25 μg of compound 2, (e) Al—H with 5 μg of compound 2, or (f)Al—H+a particulate TLR9 agonist:

MC58 NZ98 961-5945 UK355 599 (a) 16384 1024 16384 256 65536 (b) 327684096 8192 2048 >65536 (c) >65536 16384 32768 4096 >65536 (d) 32768 204816384 1024 >65536 (e) >65536 4096 8192 2048 >65536 (f) >65536 8192 163842048 >65536

Thus compound 2 improves the strain coverage compared to Al—H alone.

With a wider panel of 17 strains the percentage coverage withtiters >1024 or >4096 was as follows, with the best coverage seen usingAl—H+compound 2:

(a) (b) (c) (f) >4096 ~20% ~60% ~75% ~50% >1024 ~45% ~80% >95% ~90%

The 5CVMB vaccine was also tested in CD-1 mice with TLR7 agonistcompounds 6, 67, 68 & 71.

The antigens were administered at days 0 & 14 with (a) buffer (b) theagonist compound (c) Al—H alone (d) the agonist compound+Al—H or (e) asa positive control for compound 6 only, resiquimod +Al—H. SBA titers atday 28 are as follows:

(a) (b) (c) (d) (e) 6 64 282 256 4016 1033 67 64 64 244 949 — 68 64 —244 404 — 71 64 65 244 1653 —

Thus the activity of all tested TLR7 agonist compounds is greatlyenhanced by adsorption to Al—H (group (d) better than group (b) in allcases), and vice versa (group (d) better than group (c)).

In addition to modifying the in vivo pharmacokinetics and retention ofSMIPs, in some cases adsorption to insoluble metal salts can thusimprove immunostimulatory activity.

Effect of Adsorption on Inmunogenicity—Staphylococcus aureus

The “Combo-1” vaccine from reference 39 includes a mixture of fourpolypeptides (EsxAB, Sta006, Sta011, and Hla-H35L) and this combinationis effective for immunising against S. aureus. Reference 39 testedCombo-1 with an Al—H adjuvant, and it was decided to test Al—H incombination with adsorbed compound 2. Experiments used Balb/C mice (3intramuscular injections) and considered IgG titers, T cell responsesand protective efficacy.

FIG. 19 shows IgG titers against the individual polypeptides in Combo-1.For all four polypeptides the titer obtained using the Al—H/compound 2adjuvant combination was higher than the titer obtained using Al—H alone(**, p<0.05). Similar results were seen using Al—H with 1, 5, 25 or 50μg of compound 2.

To evaluate the quality of recall-specific T cell responses, spleencells from the mice were stimulated with the Combo-1 polypeptides.Cytokine production in CD4′ T helper cells was assessed by looking atcytokine release. The use of Al—H/compound 2 gave more antigen-specificCD4′ T cells that produce TNF-α, IL-2 and IFN-γ compared to immunisationwith unadjuvanted antigens or with antigens adjuvanted with Al—H alone.The percentage of antigen-specific CD4 T cells that produce IL-4 andIL-13 was higher (although not statistically significant) when usingAl—H compared to unadjuvanted Combo-1, but immunization using theAl—H/compound 2 combination reduced this effect at all doses except thelowest, indicating that the Th2-polarizing effect of Al—H wascounterbalanced by the Th1-polarizing effect of the TLR7 agonist.

Protective efficacy against S. aureus was assessed using a sepsis model,monitored for 15 days after challenge with Newman strain. Results fromtwo experiments with 10 μg of each polypeptide were pooled. Theproportion of animals surviving after 15 days, and the median survivallength, were:

Antigen Adjuvant % survival Survival days — — 0 1 — Al—H 4 1 — Al—H + 50μg cmpd 2 12.5 1 Combo-1 — 33.5 7 Combo-1 Al—H 21 7.5 Combo-1 Al—H + 50μg cmpd 2 97 15 Combo-1 Al—H + 25 μg cmpd 2 75 15 Combo-1 Al—H + 5 μgcmpd 2 67 15 Combo-1 Al—H + 1 μg cmpd 2 66.5 15

Survival was better, with statistical significance, when using theAl—H/compound 2 combination.

The positive effects on meningococcus B immunogenicity are thereforealso seen with S. aureus.

Effect of Adsorption on Immunogenicity—Viral Antigens

Trimeric F glycoprotein of respiratory syncytial virus (RSV) isformulated with various adjuvants, including compounds 2, 5 and example161 from reference 4 (25 μg or 100 μg per dose), with or without Al—H.Balb/C mice (6 per group) are immunized at days 0 and 21 and immuneresponses are assessed at days 35 and 42. The formulation was welltolerated in various test animals. FIG. 18 shows F-specific IgG titers(GMT, 6 mice per group) 3 weeks after a single dose of 2 μg trimeric Fprotein. Neutralization titers at day 35 are shown in FIG. 15, includingcomparisons to other adjuvants. Compound 2 plus Al—H increased bothtotal IgG titers and neutralization titers compared to Al—H alone, andthe enhancement was higher than that observed for other TLR agonists.

TLR7 agonists disclosed above were also useful for enhancing theimmunogenicity of HIV gp120 when adsorbed to Al—H and then administeredto monkeys. Results were better than with Al—H alone.

Compound 2 (10 μg/dose) was adsorbed to Al—H (100 μg Al⁺⁺⁺/dose) andtested as an adjuvant with o-gp140ΔV2 from South African HIV subtype Cstrain TV1. Immunisations used 10 μg protein in BALB/c mice (5 pergroup) on days 0 & 21 (50 μl in a single quad muscle). Other adjuvantswere also tested. FIG. 26 shows that the best results were obtainedusing compound 2 adsorbed to Al—H.

Al—H was used to adjuvant influenza virus hemagglutinin, with or withoutadsorbed compound 2. Animals received 2 doses of adjuvanted antigen. Asshown in FIG. 17A, total IgG titers were similar in the two groups afterthe first dose, but after the second dose the presence of compound 2 ledto higher titers. In addition, FIG. 17B shows that Al—H alone favouredan IgG1 isotype response (Th2-type) whereas the addition of compound 2led to a balanced IgG1/gG2a response (Th1/Th2).

The positive effects on immunogenicity with bacterial antigens aretherefore also seen with viruses.

Comparison with Other Adjuvants

The INFANRIX HEXA™ product from GlaxoSmithKline contains ≧301Udiphtheria toxoid, >401U tetanus toxoid, an acellular pertussiscomponent (25/25/8 μg of PT/FHA/pertactin), 10 μg HBsAg, a trivalent IPVcomponent (40/8/32 DU of types 1/2/3), and 10 μg Hib conjugate. Thevaccine is presented as a 5-valent aqueous vaccine which is used toreconstitute the Hib conjugate from its lyophilised form, to give a 0.5ml aqueous unit dose for human infants which contains 0.95 mg aluminiumhydroxide and 1.45 mg aluminium phosphate.

To investigate alternative adjuvants a 6-valent mixture was adjuvantedwith Al—H alone (2 mg/ml, in histidine buffer), with Al—H adsorbed tocompound 1 (1 mg/ml), or with the MF59 oil-in-water emulsion (mixed atequal volume with antigens). An adjuvant-free control was also prepared.Antigen concentrations were as follows (per ml):

DT TT PT FHA Pertactin 36.9 Lf 14.8 Lf 36.9 μg 36.9 μg 11.8 μg IPV Type1 IPV Type 2 IPV Type 3 HBsAg Hib 59.1 DU 11.8 DU 47.3 DU 14.8 μg 14.8μg

The same adjuvants were also used with a 3-valent D-T-Pa mixture (sameconcentrations).

Osmolarity and pH were measured after combining the components in orderto ensure physiological acceptability. For all 3-valent compositions thepH was between 6.2 and 7.1 and osmolarity was between 290-320 mOsm/kg.For all 6-valent compositions the pH was between 5.5 and 6.8 andosmolarity was between 260-320 mOsm/kg. A buffer control had pH 7.3 and276 mOsm/kg.

The integrity and immunogenicity of the combined antigens were alsotested. None of antigens showed an altered analytical profile afterbeing formulated as combinations i.e. the antigens and adjuvants arephysically compatible together.

With Al—H alone all antigens adsorbed well to the adjuvant. With thecomplex of Al—H+compound 1 all antigens adsorbed well, except thatpertactin was partially desorbed.

Mice (female Balb/c, 4 weeks old) were immunised intramuscularly with100 μl of each composition (i.e. 1/5 human dose) at days 0 and 28. Serawere collected 14 days after each injection. After the secondimmunisation IgG antibody titers were as follows:

No adjuvant Al—H MF59 Al—H/cmpd1 Infanrix-6 3-valent vaccines DT 75021626 15693 23395 — TT 13120 17868 22458 23131 — Pertactin 639 720910258 12857 — PT 2501 8270 7212 9938 — FHA 3982 12057 14098 23008 —6-valent vaccine DT 1751 18914 13982 23102 21581 TT 12729 16756 2222923267 15998 Pertactin 333 6299 9363 5153 10809 PT 3069 3384 4823 64846052 FHA 4558 7206 16201 19383 11051 Hib 177 813 1266 2153 1269 HBsAg1058 1598 2288 4501 1113

Thus for all of these antigens the inclusion of an adjuvant increasedIgG antibody titers. The best titers were seen when using the complex ofAl-h and compound 1. The next best were with MF59, which gave betterresults than aluminium hydroxide alone. The titers obtained using theadsorbed complex were better for all antigens than those seen withInfanrix Hexa, except for pertactin.

Furthermore, the data show that the good results achieved with the3-valent vaccine are maintained even after IPV, Hib and HBsAg are added.

IgG responses were also investigated by subclass. For most of theantigens in the 6-valent vaccines the adjuvants had little effect onIgG1 titers, but they did increase IgG2a and IgG2b titers. The bestIgG2a and IgG2b titers were obtained with the adsorbed complex, and thenwith MF59.

The increased titers seen with the complex compared with Al—H alone, orwith the mixture of aluminium salts seen in Infanrix Hexa™, mean thatthe total amount of aluminium per dose can be reduced while maintainingenhancement of immune responses.

Reduction of Antigen Doses

Experiments were designed to investigate whether the adjuvant complexcould be used to reduce the amount of antigen per dose. 10-fold, 50-foldand 100-fold dilutions (relative to human dosing i.e. to deliver 1 μg,0.2 μg or 0.1 μg HBsAg to each mouse per 100 μl dose) of the 6-valentantigen combinations were made while adjuvant concentration wasmaintained.

For all 6-valent compositions the pH was between 6.1 and 7.0 andosmolarity was between 275-320 mOsm/kg. A buffer control had pH 7.3 and285 mOsm/kg.

Mice were immunised in the same way as discussed above. Total serum IgGtiters after 2 immunisations were as follows:

No adjuvant Al—H MF59 Al—H + cmpnd 1 1/10 1/50 1/100 1/10 1/50 1/1001/10 1/50 1/100 1/10 1/50 1/100 DT 459 2043 137 18357 13106 7541 174316003 8736 21913 16807 13724 TT 7602 7929 1700 17595 9664 5531 2279112062 13015 23570 12237 13183 Pertactin 827 2154 341 10880 8135 418117159 10591 7288 17098 10748 8952 PT 3612 5645 2129 5287 3266 1068 72003659 5493 9051 4203 2717 FHA 2305 4161 101 8997 4471 1442 19197 51794492 22151 8293 3252 Hib 171 352 109 1380 796 251 3147 573 2415 30561440 1815 HBsAg 525 412 129 1034 685 226 4885 1103 1983 5270 1526 950

Thus the presence of adjuvants allowed a dose reduction of 5-fold or10-fold while maintaining IgG titers which are comparable or higher tounadjuvanted antigens. MF59 and the adsorbed complex in particular areuseful for dose sparing of antigens in this manner.

Adjuvant Dosing

With the 100-fold antigen dilution the amount of adjuvant was alsoreduced. The complex of Al—H and compound 1 was prepared at 3 strengthshaving 2 mg/ml Al—H with either 5 μg, 25 μg or 100 μg of compound 1 perdose. For comparison a 1:100 antigen dose was tested in unadjuvantedform or with Al—H alone. A 1:100 dilution of INFANRIX HEXA was also usedfor comparison.

For all 6-valent compositions the pH was between 6.2 and 7.3 andosmolarity was between 270-320 mOsm/kg. A buffer control had pH 7.3 and280 mOsm/kg.

Mice were immunised as before. Total serum IgG titers after 2immunisations were as follows:

Infanrix Al—H + compound 1 No adjuvant Hexa Al—H 100 25 5 DT 584 628210849 21571 20865 11788 TT 3426 5415 6857 16041 15124 6236 Pertactin 483017 6053 6158 6697 3815 PT 3351 1751 2699 2476 2696 3079 FHA 262 78865626 7369 8634 6120 Hib 126 109 310 936 792 390 HBsAg 88 240 369 40622308 1154

Thus lower amounts of the complex still retain good adjuvanticity andcan induce higher IgG antibody titers than those induced by unadjuvanted6-valent antigen formulations. By reducing the amount of adjuvant, whilemaintaining immunological efficacy, the safety profile of a vaccine canbe improved which is particularly important in pediatric settings.

Adjuvant Dilution

Compound 2 was adsorbed to Al—H as disclosed above. This adsorbedmaterial was then mixed with plain Al—H. Flow cytometry showed a shiftof fluorescence for the entire Al—H population, indicatingredistribution of the adsorbed compound onto the new Al—H.

To study the potential for redistribution a two-chamber experiment wasprepared. The two chambers were separated by a membrane with a 8-10 kDacutoff. In a control experiment Al—H was placed in chamber 1 and thesame volume of PBS was placed in chamber 2. No leakage of Al—H fromchamber 1 into chamber 2 was observed. In the test experiment compound2+Al—H was placed in chamber 1 and the same volume of Al—H was placed inchamber 2. Flow cytometry, based on the intrinsic fluorescence ofcompound 2, showed that the compound had crossed from chamber 1 tochamber 2.

Similar experiments were performed with phosphorylated hexaacyldisaccharide, a synthetic analog of monophosphoryl lipid A (MPL), usingNMR. The experiments showed that MPL can pass through the membrane fromchamber 1, and adsorb to Al—H in chamber 2.

Thus a bulk mixture of Al—H and an adsorbed SMIP can be prepared at ahigh SMIP concentration, and this bulk can be diluted with plain Al—H togive a desired SMIP strength. This arrangement can simplify manufactureof several different strengths of day-to-day adjuvant from a singlebulk.

Extemporaneous Mixing of Antigen and Adjuvant

The modified 5CVMB combination was prepared in lyophilised form [92]with 10% sucrose, 3.84 mg/ml NaCl and 10 mM phosphate buffer (pH 7.0).This material was reconstituted with an aqueous adjuvant component with500 μg/ml of compound 2, 3 mg/ml Al—H, 2% sucrose, 6.25 mg/ml NaCl and10 mM histidine buffer (pH 6.3).

This material was compared to an aqueous formulation of the samematerials (500 μg/ml compound 2, 10 mM histidine buffer pH 6.3, 6.25mg/ml NaCl, 2% sucrose, 3 mg/ml Al—H).

The TLR7 agonist remained adsorbed to Al—H after mixing with thelyophilised antigens.

Antigen adsorption was checked after mixing (time 0) and after 24 hoursof storage at 2-8° C. or at room temperature. Antigen adsorption wascomparable in the lyophilised/reconstituted formulation and in thefully-aqueous formulation. Both formulations were stable for 24 hours.

Thus antigens and adjuvant can be stored separately for extemporaneousmixing at the point of use.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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What is claimed:
 1. A TLR agonist selected from the group consisting of(a) a TLR4 agonist comprising at least one adsorptive moiety, whereinthe TLR agonist has a molecular weight of less than 1000 Da; (b) a TLR4agonist comprising at least one adsorptive moiety, wherein the TLRagonist has a molecular weight of less than 1000 Da. aTLR4 agonistcomprising at least one adsorptive moiety, provided that the TLR4agonist does not include an acyl chain; (c) a TLR9 agonist comprising atleast one adsorptive moiety, provided that the TLR9 agonist does notinclude a cytidine nucleoside, (d) a TLR agonist including between twoand fifteen adsorptive moieties, and (e) a TLR2 agonist comprising atleast one adsorptive moiety, provided that the TLR2 agonist is not acompound according to formula (III)

wherein: R¹ is H, —C(O)—C₁₀-C₁₈alkyl; R² is C₁₀-C₁₈alkyl; R³ isC₁₀-C₁₈alkyl; L₁ is —CH₂O—, —CH₂OC(O)—, —CH₂NR⁷C(O)— or —C(O)NR⁷—; L₂ is—O—, —OC(O)— or —NR⁷C(O)—; R⁴ is -L₃R⁵ or -L₄R⁵; R⁵ is —N(R⁷)₂, —OR⁷,—P(O)(OR⁷)₂, —C(O)OR⁷, —NR⁷C(O)L₃R⁸, —OL₃R⁶, —C(O)NR⁷L₃R⁸, C₁-C₆alkyl, aC₆aryl, a C₁₀aryl, a C₁₄aryl, 5 to 14 membered heteroaryl containing 1to 3 heteroatoms selected from O, S and N, C₃-C₈cycloalkyl or a 5 to 6membered heterocycloalkyl containing 1 to 3 heteroatoms selected from O,S and N, wherein the aryl, heteroaryl, cycloalkyl and heterocycloalkylof R⁵ are each optionally substituted with 1 to 3 substituentsindependently selected from —OR⁹, —OL₃R⁶, —OL₄R⁶, —OR⁷, and —C(O)OR⁷; L₃is a C₁-C₁₀alkylene, wherein the C₁-C₆alkylene of L₃ is optionallysubstituted with 1 to 4 R⁶ groups, or the C₁-C₆alkylene of L₃ issubstituted with 2 C₁-C₆alkyl groups on the same carbon atom whichtogether, along with the carbon atom they are attached to, form aC₃-C₈cycloakyl; L₄ is —((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)— or—(CR¹¹R¹¹)((CR⁷R⁷)_(p)O)_(q)(CR¹⁰R¹⁰)_(p)—, wherein each R¹¹ is aC₁-C₆alkyl groups which together, along with the carbon atom they areattached to, form a C₃-C₈cycloakyl; each R⁶ is independently selectedfrom halo, C₁-C₆alkyl, —OR⁷, —N(R⁷)₂, —C(O)N(R⁷)₂, —P(O)(OR⁷)₂, a C₆aryl, a C₁₀ aryl and a C₁₄ aryl; each R⁷ is independently selected fromH and C₁-C₆alkyl; R⁸ is selected from —SR⁷, —C(O)OH and a 5 to 6membered heterocycloalkyl containing 1 to 3 heteroatoms selected from Oand N; R⁹ is phenyl; each R¹⁰ is independently selected from H and halo;each p is independently selected from 1, 2, 3, 4, 5 and 6, and q is 1,2, 3 or
 4. 2. A TLR7 agonist selected from compounds according to any offormulae (D), (E) or (H):

wherein: (a) P³ is selected from H, C₁-C₆alkyl, CF₃, and—((CH₂)_(p)O)_(q)(CH₂)_(p)O_(s)— and —Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁴is selected from H, C₁-C₆alkyl, —C₁-C₆alkylaryl and—Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one of P³and P⁴ is —Y-L-X—P(O)(OR^(X))(OR^(Y)), (b) P⁵ is selected from H,C₁-C₆alkyl, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P⁶ is selected from H,C₁-C₆alkyl each optionally substituted with 1 to 3 substituents selectedfrom C₁-C₄alkyl and OH, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁷ isselected from H, C₁-C₆alkyl, —((CH₂)_(p)O)_(q)(CH₂)_(p)O—, —NHC₁-C₆alkyland —Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one ofP⁵, P⁶ and P⁷ is —Y-L-X—P(O)(OR^(X))(OR^(Y)); (c) P⁸ is selected from H,C₁-C₆alkyl, C₁-C₆alkoxy, —NHC₁-C₆alkyl each optionally substituted withOH, and —Y-L-X—P(O)(OR^(X))(OR^(Y)); and P⁹ and P¹⁰ are eachindependently selected from H, C₁-C₆alkyl, C₁-C₆alkoxy, —NHC₁-C₆alkyleach optionally substituted with OH and C₁-C₆alkyl, and—Y-L-X—P(O)(OR^(X))(OR^(Y)); with the proviso that at least one of P, P⁹or P¹⁰ is —Y-L-X—P(O)(OR^(X))(OR^(Y)); (d) P¹⁶ and each P¹⁸ are eachindependently selected from H, C₁-C₆alkyl, and—Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹⁷ is selected from H, C₁-C₆alkyl, aryl,heteroaryl, C₁-C₆alkylaryl, C₁-C₆alkyl heteroaryl,C₁-C₆alkylaryl-Y-L-X—P(O)(OR^(X))(OR^(Y)) and—Y-L-X—P(O)(OR^(X))(OR^(Y)), each optionally substituted with 1 to 2substituents selected from C₁-C₆alkyl or heterocyclyl with the provisothat at least one of P¹⁶, P¹⁷ or a P¹⁸ contains a—Y-L-X—P(O)(OR^(X))(OR^(Y)) moiety; R^(X) and R^(Y) are independentlyselected from H and C₁-C₆alkyl; R^(C), R^(D) and R^(H) are eachindependently selected from H and C₁-C₆alkyl; X^(C) is selected from CHand N; R^(E) is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy,C(O)C₁-C₆alkyl, halogen and —((CH₂)_(p)O)_(q)(CH₂)_(p)—; X^(E) isselected from a covalent bond, CR^(E1)R^(E3) and NR^(E4); R^(E2), R^(E3)and R^(E4) are independently selected from H and C₁-C₆alkyl;X^(H1)-X^(H2) is selected from —CR^(H2)R^(H3)—,CR^(H2)R^(H3)—CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—, —C(O)CR^(H2)R^(H3)—,—CR^(H2)R^(H3)C(O)—, —NR^(H4)C(O)—, C(O)NR^(H4)—, CR^(H2)R^(H3)S(O)₂ and—CR^(H2)═CR^(H2)—; R^(H2), R^(H3) and R^(H4) are each independentlyselected from H, C₁-C₆alkyl and P¹⁸; X^(H3) is selected from N and CN; Xis selected from a covalent bond, O and NH; Y is selected from acovalent bond, O, C(O), S and NH; L is selected from, a covalent bondC₁-C₆alkylene, C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxyand —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4substituents independently selected from halo, OH, C₁-C₄alkyl,—OP(O)(OH)₂ and —P(O)(OH)₂; m is selected from 0 or 1; each p isindependently selected from 1, 2, 3, 4, 5 and 6; q is selected from 1,2, 3 and 4; and s is selected from 0 and 1
 3. A compound of formula (F):

wherein: R^(X) and R^(Y) are independently selected from H andC₁-C₆alkyl; Q and V are independently selected from a covalent bond, NH,S, C(O) and O; X is selected from a covalent bond, O and NH; Y isselected from a covalent bond, O, C(O), S and NH; L is selected from, acovalent bond C₁-C₆alkylene, C₁-C₆alkenylene, arylene, heteroarylene,C₁-C₆alkyleneoxy and —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionallysubstituted with 1 to 4 substituents independently selected from halo,OH, C₁-C₄alkyl, —OP(O)(OH)₂; and —P(O)(OH)₂; and R^(F1), R^(F2) andR^(F3) are independently selected from H, C₁-C₁₈ alkyl, C₁-C₁₈alkenyl,C(O)C₁-C₁₈alkyl and C(O)C₁-C₁₈alkenyl; each p is independently selectedfrom 1, 2, 3, 4, 5 and 6; and q is selected from 1, 2, 3 and
 4. 4. Acompound of formula (G)

wherein: P¹¹ is selected from H, C₁-C₆alkyl, C₁-C₆alkoxy, NR^(V)R^(W)and —Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹² is selected from H, C₁-C₆alkyl,aryl optionally substituted by —C(O)NR^(V)R^(W), and—Y-L-X—P(O)(OR^(X))(OR^(Y)); P¹³, P¹⁴ and P¹⁵ are independently selectedfrom H, C₁-C₆alkyl, C₁-C₆ alkoxy and —Y-L-X—P(O)(OR^(X))(OR^(Y)); withthe proviso that at least one of P¹¹, P¹², P¹³, P¹⁴ or P¹⁵ is—Y-L-X—P(O)(OR^(X))(OR^(Y)); R^(V) and R^(W) are independently selectedfrom H, C₁-C₆alkyl or together with the nitrogen atom to which they areattached form a 4 to 7 remembered heterocyclic ring; X^(G) is selectedfrom C, CH and N;

represents an optional double bond, wherein X^(G) is C if

is a double bond; and R^(G) is selected from H and C₁-C₆alkyl; X isselected from a covalent bond, O and NH; Y is selected from a covalentbond, O, C(O), S and NH; L is selected from, a covalent bondC₁-C₆alkylene, C₁-C₆alkenylene, arylene, heteroarylene, C₁-C₆alkyleneoxyand —((CH₂)_(p)O)_(q)(CH₂)_(p)— each optionally substituted with 1 to 4substituents independently selected from halo, OH, C₁-C₄alkyl,—OP(O)(OH)₂ and —P(O)(OH)₂; each p is independently selected from 1, 2,3, 4, 5 and 6 and q is selected from 1, 2, 3 and
 4. 5. A compositioncomprising the TLR agonist of claim 1 and an insoluble metal salt. 6.The composition of claim 5, wherein the TLR agonist is adsorbed onto theinsoluble metal salt.
 7. The composition of claim 6, wherein at least80% of the total TLR agonist in the composition is adsorbed ontoinsoluble metal salt.
 8. The composition of claim 7, wherein theadsorbed TLR agonist is, when injected intramuscularly, still present inthe injected muscle at least 12 hours later.
 9. The composition of claim7, wherein the adsorbed TLR agonist, when injected intramuscularly, hasa lower peak serum concentration (Cmax) than the same TLR agonist wheninjected intramuscularly in non-adsorbed form.
 10. A process forpreparing an adjuvant complex, comprising a step of mixing the TLRagonist of claim 1 with an insoluble metal salt such that the TLRagonist interact to form the complex.
 11. The process of claim 10,further comprising (ii) sterilising the complex.
 12. A process forpreparing an immunogenic composition, wherein the process comprisesmixing the TLR agonist of claim 1, an insoluble metal salt, and animmunogen, thereby providing the immunogenic composition.
 13. Acomposition comprising: (a) an adjuvant complex prepared by the processof claim 10; and (b) at least two different immunogens.
 14. Thecomposition of claim 13, wherein the adjuvant complex comprises the TLRagonist adsorbed to the insoluble metal salt.
 15. The composition ofclaim 13, wherein the composition includes (i) a diphtheria toxoid, atetanus toxoid, and an acellular pertussis antigen; (ii) a diphtheriatoxoid, a tetanus toxoid, a pertussis antigen, and a H. influenzae typeB capsular saccharide conjugate; (iii) a diphtheria toxoid, a tetanustoxoid, a pertussis antigen, and a hepatitis B virus surface antigen;(iv) a diphtheria toxoid, a tetanus toxoid, a pertussis antigen, ahepatitis B virus surface antigen and a H. influenzae type B capsularsaccharide conjugate; (v) a diphtheria toxoid, a tetanus toxoid, apertussis antigen, and an inactivated poliovirus antigen; or (vi) adiphtheria toxoid, a tetanus toxoid, a pertussis antigen, a H.influenzae type B capsular saccharide conjugate, a hepatitis B virussurface antigen, and an inactivated poliovirus antigen.
 16. Thecomposition of claim 15, wherein the composition also includes furtherantigens, such as from N. meningitidis or S. pneumoniae.
 17. Thecomposition of claim 13, wherein the composition includes at least onebacterial antigen and at least one viral antigen.